to my parents for their unyielding support and encouragement
TRANSCRIPT
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EVALUATION OF BACILLUS SUBTILIS R0179 ON GENERAL WELLNESS, GASTROINTESTINAL SYMPTOMS AND GASTROINTESTINAL VIABILITY: A
RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED, CLINICAL TRIAL IN HEALTHY ADULTS
By
ABDULAH HANIFI
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2013
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© 2013 Abdulah Hanifi
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To my parents for their unyielding support and encouragement
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ACKNOWLEDGMENTS
I would like to thank my advisor and mentor Dr. Wendy Dahl whose knowledge
and guidance has invariably led to my success in a field of science that I have now
grown to love. Thank you Dr. Dahl for taking a chance on me at a time when I was lost
in a sea of opportunity. I would also like to extend the most sincere gratitude to my
colleagues: Dr. Tyler Culpepper for his support and guidance in the field of
microbiology, Amanda Ford for her invaluable aid, advice and willingness to help me
learn what it takes to run a clinical trial, Lauren Khouri for her continued patience and
willingness to help, Allyson Radford for her expertise and guidance in regulatory affairs
and Dr. Younis Salmean for his sage wisdom in navigating the currents of graduate
studies. I would also like to thank the study volunteers who dedicated some of their
valuable time towards helping me achieve my graduate degree.
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TABLE OF CONTENTS Page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 8
LIST OF FIGURES .......................................................................................................... 9
LIST OF ABBREVIATIONS ........................................................................................... 10
ABSTRACT ................................................................................................................... 11
CHAPTER
1 LITERATURE REVIEW .......................................................................................... 13
Introduction to the Intestinal Microbiome ................................................................ 13
Origins of the Probiotic Concept ............................................................................. 15 Modern Definition of Probiotics ............................................................................... 16
Identification and Selection of Probiotic Strains ...................................................... 16 Technical Aspects ............................................................................................ 17
Physiological Aspects ....................................................................................... 18 Functional Aspects ........................................................................................... 19
Evaluating the Safety of Probiotic Bacteria ............................................................. 20 Guidelines for Evaluating the Safety of Bacterial Strains.................................. 20
Systemic infections .................................................................................... 21 Deleterious metabolic activity ..................................................................... 21
Gene transfer ............................................................................................. 22 Excessive immune stimulation ................................................................... 22
In Vitro and Animal Studies .............................................................................. 23 Clinical Trials .................................................................................................... 24
Quality of Life Assessment ..................................................................................... 25 Development of the GSRS ............................................................................... 25
Initial Validation of the GSRS ........................................................................... 26 An Updated GSRS ........................................................................................... 27
Internal consistency reliability .................................................................... 28 Construct validity ........................................................................................ 29
Discriminant validity ................................................................................... 30 Responsiveness ......................................................................................... 31
Determination of Healthy Status in Research Participants ...................................... 32 Bacillus subtilis as a Probiotic ................................................................................. 35
In Vitro Studies ....................................................................................................... 41 The Safety of Bacillus subtilis ........................................................................... 41
The Safety of Bacillus subtilis R0179 ............................................................... 42 Animal Studies ........................................................................................................ 43
Germination of Bacillus subtilis in the Gastrointestinal Tract ............................ 43 B. subtilis in the Human Intestinal Tract ........................................................... 46
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Potential Health Benefits of Bacillus subtilis ........................................................... 47 The Effect of B. subtilis on Immune Function ................................................... 47
The Effect of B. subtilis on Bone Metabolism ................................................... 49 The Use of B. subtilis as Bowel Preparation for Colonoscopy .......................... 50
Conclusions ............................................................................................................ 51
2 PURPOSE .............................................................................................................. 53
3 METHODS AND PROCEDURES ........................................................................... 54
Study Design .......................................................................................................... 54
Inclusion and Exclusion Criteria before Attaining Consent...................................... 54 Recruitment ............................................................................................................ 55
Baseline .................................................................................................................. 56 Randomization and Intervention ............................................................................. 57
Washout and Post Intervention ............................................................................... 58 Compensation ......................................................................................................... 58
Stool Protocol ......................................................................................................... 58 Statistical Methods .................................................................................................. 59
Equivalence Testing ......................................................................................... 59 B. subtilis Viability ............................................................................................. 61
4 RESULTS ............................................................................................................... 65
Participants ............................................................................................................. 65
Daily Questionnaire Analysis .................................................................................. 65 Gastrointestinal Symptom Rating Scale Analysis ................................................... 69
Viability of B. subtilis R0179 in Humans.................................................................. 70
5 DISCUSSION AND CONCLUSIONS ...................................................................... 96
Discussion .............................................................................................................. 96 Conclusions .......................................................................................................... 100
APPENDIX
A IRB APPROVAL ................................................................................................... 101
B INFORMED CONSENT ........................................................................................ 102
C GLOBAL PHYSICAL ACTIVITY QUESTIONNNAIRE........................................... 112
D DAILY QUESTIONNAIRE ..................................................................................... 115
E GASTROINTESTINAL SYMPTOM RESPONSE SCALE ..................................... 118
LIST OF REFERENCES ............................................................................................. 120
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BIOGRAPHICAL SKETCH .......................................................................................... 126
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LIST OF TABLES
Table page 1-1 Syndrome assignments of the SF-36, PGWB, and GSRS ................................. 52
3-1 Daily Questionnaire syndrome assignments....................................................... 63
3-2 Gastrointestinal Symptom Rating Scale syndrome assignments........................ 64
4-1 Participant demographics ................................................................................... 72
4-2 Daily Questionnaire syndrome equivalence testing ............................................ 73
4-3 Daily Questionnaire symptom equivalence testing ............................................. 82
4-4 Gastrointestinal Symptom Rating Scale equivalence testing .............................. 89
4-5 Gastrointestinal viability of Bacillus spores ......................................................... 95
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LIST OF FIGURES
Figure page 3-1 Study design ....................................................................................................... 62
4-1 Participant flow diagram ..................................................................................... 71
4-2 Gastrointestinal distress syndrome ..................................................................... 76
4-3 Cephalic syndrome ............................................................................................. 77
4-4 Epidermal syndrome ........................................................................................... 78
4-5 Ear nose and throat syndrome ........................................................................... 79
4-6 Behavioral syndrome .......................................................................................... 80
4-7 Emetic syndrome ................................................................................................ 81
4-8 Constipation symptom ........................................................................................ 84
4-9 Diarrhea symptom .............................................................................................. 85
4-10 Fatigue symptom ................................................................................................ 86
4-11 Bowel movement frequency ............................................................................... 87
4-12 Hours of sleep .................................................................................................... 88
4-13 GSRS abdominal pain syndrome ....................................................................... 90
4-14 GSRS reflux syndrome ....................................................................................... 91
4-15 GSRS indigestion syndrome............................................................................... 92
4-16 GSRS constipation syndrome............................................................................. 93
4-17 GSRS diarrhea syndrome................................................................................... 94
4-18 Gastrointestinal viability ...................................................................................... 95
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LIST OF ABBREVIATIONS
CFU Colony Forming Units DQ Daily Questionnaire EFSA European Food Safety Administration
FAO Food and Agricultural Organization
FDA Food and Drug Administration GERD Gastroesphogeal reflux disease GI Gastrointestinal GPAQ Global Physical Activity Questionnaire GRAS Generally recognized as safe GSRS Gastrointestinal Symptom Rating Scale
IBS Irritable Bowel Syndrome
ICC Intraclass coefficients PBS Phosphate buffered saline PGWB Psychological general well-being index QPS Qualified Presumption of Safety
SF-36 Short Form 36
SP Species
WHO World Health Organization
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
EVALUATION OF BACILLUS SUBTILIS R0179 ON GENERAL WELLNESS, GASTROINTESTINAL SYMPTOMS AND GASTROINTESTINAL VIABILITY: A
RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED, CLINICAL TRIAL IN HEALTHY ADULTS
By
Abdulah Hanifi
December 2013
Chair: Wendy Dahl Major: Food Science and Human Nutrition
Bacillus subtilis R0179 has been marketed in Asia as the probiotic Medilac®.
This probiotic formulation is composed of Enterococcus faecium R0026 and Bacillus
subtilis R0179 in a 9:1 ratio. However, prior research has not evaluated B. subtilis
R0179 as a single probiotic agent at doses higher than 0.1 billion CFU/capsule. To
confirm the oral dose-response tolerance of B. subtilis R0179, a 6-week randomized,
double-blind, placebo-controlled clinical trial in healthy adults (n=81), 18-50 years of
age, was conducted. Methods: Participants were randomized to one of three B. subtilis
R0179 dosages (0.1, 1 and 10 billion cfu/capsule/day) or placebo for a 4-week
intervention period. General wellness was evaluated using a daily questionnaire (DQ)
composed of six syndromes rated on a scale of 0 (none) to 6 (very severe):
gastrointestinal distress, cephalic, ear-nose-throat, behavioral, emetic, and epidermal.
Gastrointestinal symptoms were evaluated pre-intervention, during intervention and post
intervention with the Gastrointestinal Symptom Rating Scale (GSRS). The GSRS
contains five syndromes rated on a scale of 1 (no discomfort) to 7 (very severe
discomfort): abdominal pain, indigestion, reflux, constipation and diarrhea. Three stool
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samples were obtained, at pre-intervention, intervention and post intervention time
points to assess the viability of B. subtilis in the human gastrointestinal tract. Symptom
data were analyzed using the equivalence testing method to assess dose response by
week. Kruskall-Wallis tests were used to compare pre-intervention and intervention
syndrome scores for both the GSRS and DQ. Gastrointestinal viability was compared
on a per week by treatment basis using ANOVA and Tukey-Kramer HSD when
appropriate. Results: General wellness and gastrointestinal function was not adversely
affected by oral consumption of B. subtilis R0179 at any dose. Mean intervention DQ
syndromes were not different from baseline and ranged from 0.04 to 0.7. Mean
intervention GSRS syndromes were not different from baseline and ranged from 1.1 to
1.9. Fecal viable counts of B. subtilis R0179 demonstrated a dose response ranging
from 101 to 105 CFU/g. No side effects of any kind were reported. Conclusions: Overall,
the results demonstrate that consumption of B. subtilis R0179 was well tolerated by
healthy human adults and is viable in the human gastrointestinal tract.
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CHAPTER 1 LITERATURE REVIEW
Introduction to the Intestinal Microbiome
The human gastrointestinal tract is home to a large number of microbes which
first colonize humans during the fetal stage of development. This colonization process
continues during birth and up through adolescence (1, 2). The composition of these
microbes varies from person to person and is impacted by personal and environmental
factors such as the decision to breast or formula feed an infant (3) and the nosocomial
spread of microbes in maternity wards (4). During late adolescence the intestinal
microbiome begins to stabilize in both number and composition (5). In healthy adults,
the general intestinal microbiome is stable and remains so until old age when a decline
in gastrointestinal function begins to cause a shift in the number and variety of species
(6).
The variation of microbial species in the human gastrointestinal tract differs from
one individual to another but overall, nearly 97% of gut microbes are strict anaerobes
while the remaining 3% are aerobic. These consist of autochthonous species
(permanent residents) and allochothonous species (transitory colonizers) that make up
the 400 to 1000 species present throughout the gastrointestinal tract (7). Autochthonous
species are typically acquired in the early years of human life while the allochothonous
species enter the gastrointestinal tract through activities like ingestion of food.
Allochothonous species temporarily colonize the gastrointestinal tract and are
subsequently excreted along with feces. The identification of these microbes and the
roles they play in healthy gastrointestinal function is consistently being evaluated with
international projects such as the US National Institutes of Health’s Human Microbiome
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Project, the European Commission’s Metagenomics of the Human Intestinal Tract
project and the Canadian Microbiome Initiative (8). These projects have provided great
insight into the importance of gastrointestinal microbes and the association of certain
disease states to differing microbiome compositions. However, a conclusive definition of
a healthy human microbiome is not yet defined.
Current research has only just begun to piece together the important components
of a healthy microbiome. A majority of microbiota research has focused on
understanding the composition of the intestinal microbiome in sick or diseased
individuals and then comparing them to controls. The main difference identified between
a healthy and an unhealthy microbiome is the high and low diversity of species,
respectively (9). Recent studies have demonstrated a link between low diversity in the
human intestinal microbiome and both obesity and inflammatory bowel disease (10, 11).
These disease states are a result of the inability of the gastrointestinal bacteria to resist
ecologic stressors like pathogenic bacteria and antibiotic usage (8).
Ecologic stressors cause temporary imbalances of the intestinal microbiome and
can either have a negative impact (dysbiosis) or a positive impact (probiosis) on the
function of the gut. For example, antibiotic usage is regarded as a dysbiotic event
because of its ability to reduce the number and composition of autochthonous
organisms in the gut which increases the chances of pathogenic organisms to take up
residence in the gut (12). Disease states like antibiotic-associated diarrhea, metabolic
syndrome and atopic eczema have been associated with dysbiosis of the intestinal
microbiome (13). The converse is true for probiosis of the intestinal microbiome which
may serve to maintain or reestablish the homeostasis between host and microbiome.
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The current state of research has begun to enumerate the health benefits provided to
humans by their intestinal microbiome. The purpose of this literature review is first to
identify the origins of the probiotic concept, then to define modern probiotics and outline
their regulation, next to understand how research is conducted on novel probiotic
agents and finally to evaluate the usage of a spore-forming bacteria as a probiotic.
Origins of the Probiotic Concept
Elie Metchnikoff’s Prolongation of Life, published in 1907, is one of the first
substantial works evaluating dysbiosis and probiosis. In this work, he examines how
various mechanisms that modulate the intestinal microbiome can prolong life (14). In a
section titled “Intestinal Putrefaction Shortens Life”, he examines the role of bacteria in
the bowels. He begins to describe that in the first hours after birth the microbes present
in the infant’s intestinal tract thrive on shed intestinal mucosal cells. The check against
the overgrowth of these opportunistic microbes is the consumption of mother’s milk
which promotes the colonization of the infantile gastrointestinal tract with Bifidobacteria
(14). Metchnikoff then diverts his attention to the fact that because microbes cause the
natural putrefaction of food outside of the bowels they may also be the cause of the
putrefaction of the bowels. Some foods like milk, however, resist putrefaction, because
of the preservative act of souring (14). He attributes this preservation of food to the
production of lactic acid, a by-product of glucose fermentation.
Metchnikoff continues this train of thought by reviewing research that evaluated
the anti-putrefactive properties of the consumption of yoghurt derived Lactobacilli
cultures and lactic acid (14). Metchnikoff cites numerous studies where consumption of
lactic acid producing microbes decreased gastrointestinal symptoms and increased
quality of life. The research conducted was anecdotal by today’s standards but the
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conclusions that Metchnikoff draws from them are very important to history of probiotic
usage. He advocates for the consumption of live microbes and lactic acid that suppress
the naturally putrefactive intestinal microbes much in the same way that a mother’s
breast milk suppresses the overgrowth of microbes in an infant (14). These concepts of
probiosis and dysbiosis have since evolved and are continually being revised based on
scientific discoveries.
Modern Definition of Probiotics
The concept of probiosis and the probiotic effect of microbes on the
gastrointestinal tract is a heavily researched topic throughout the world. In the past 20
years, the number of research publications on intestinal microbiota has increased
fivefold with about 500 publications occurring in 2009 and almost double that occurring
in 2012 (15). The importance of this field of research has reached a global scale and so
the Food and Agriculture Organization (FAO) and the World Health organization (WHO),
have published a working group report on probiotics and their importance (16). They
define probiotics as “live microorganisms which when administered in adequate
amounts confer a health benefit on the host” (16). The identification of these
microorganisms is the first step in a process to determine the safety and efficacy of
probiotics. The report continues on to describe preferred identification and evaluation
methods of these microorganisms.
Identification and Selection of Probiotic Strains
The process of selecting a particular type of bacteria for use as a probiotic and
evaluating it for safety begins with its identification (17). The first step in identifying a
microbe is by characterizing its genus, species, strain and origin. Identifying the
bacterium in this manner also allows scientists to assess its pathogenicity by comparing
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it to genetically similar microorganisms (18). For example, if a certain species of
pathogenic bacteria typically produces enterotoxins (proteins produced by bacteria
which can cause gastrointestinal disorders like vomiting and diarrhea), and is antibiotic
resistant, then this organism would be unfit for human consumption. Similarly, if the
bacteria being identified is genetically similar to the pathogenic bacteria then it may be
ruled out as a probiotic. The gold standard by which a bacterium can be identified at the
strain specific level is through DNA-DNA hybridization (16). This technique allows the
DNA of several different species of bacteria to be compared against one another. The
identification process also includes a characterization of the bacteria’s technical,
physiological and functional aspects. These aspects act as a set of criteria to assess the
probiotic activity of the bacteria in vitro using models of the human gastrointestinal tract
(18).
Technical Aspects
The technical aspects of bacteria that are characterized are growth properties
during processing, and viability during transport and storage (18). Evaluating growth
properties in vitro allows scientists to understand the ideal growth conditions of the
bacteria while characterizing the viability of the bacteria during transport and storage
contributes to their mass marketing potential (18). If a bacteria grows readily but is
unable to survive transport and storage then its ability to cause a probiotic effect after
consumption is lost. A third technical aspect which is important to consumers are the
sensory properties of the probiotic (19). If current trends continue, more and more
probiotics will be added to foods forming “functional foods”, which provide a benefit to
the consumer beyond basic sustenance. Evaluation of the sensory properties of a
probiotic in food will contribute to its overall acceptability by consumers.
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Physiological Aspects
Physiological aspects of bacteria that are characterized are the ability to
withstand environmental stressors, and the ability of the bacteria to adhere to human
intestinal epithelial cells (18). Environmental stressors can include a lack of nutrient
availability, fluctuations in temperature, and competition among species. Upon
consumption, bacteria must be able to survive the antimicrobial properties of the human
gastrointestinal tract such as stomach acid, bile acid, and pancreatic juices (18).
The ability of the bacteria to survive the acidic conditions of the stomach and
upper gastrointestinal tract are essential for it to begin transitory colonization in the
jejunum and ileum where the majority of bacteria reside. While some colonization does
occur in the stomach and duodenum (101 and 103 respectively), a large number of
bacteria spend a part of the life cycle in the jejunum, ileum and colon (104, 107, and 1012
respectively) (15). The bacteria may also be able to digest bile salts which act as
antimicrobials in the gut, though not all probiotics possess this ability (20). Lastly, the
probiotic bacteria must withstand the various digestive enzymes found in pancreatic
juices. After being exposed to all of these environmental stressors, the bacteria must be
able to adhere to intestinal epithelial cells.
Bacterial adhesion to intestinal epithelial cells allows them to begin transitory
colonization of the lower small intestine and colon (21). The adhesion also serves to
competitively exclude other species from colonization thus increasing the chances of
survival of the bacteria (15). Probiotic bacteria that can withstand the harsh
antimicrobial conditions of the gastrointestinal tract behave just like hardy pathogenic
bacteria, and because they are able to survive, they can competitively exclude the
pathogenic bacteria from adhering to intestinal epithelial cells. This prevents dysbiosis
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from occurring. Once it has been demonstrated that a bacteria possesses the
necessary technical and physiological aspects, the functional and potential health
benefits of the bacteria are characterized (18).
Functional Aspects
Primary functional aspects of probiotics include the production of antimicrobials
and stimulation of autochthonous microbes. When considered in tandem, these aspects
serve to divest the gastrointestinal tract of pathogenic bacteria and subsequently
enhance the growth of resident gut microbes (15). The potential health benefits of
probiotic bacteria has been well documented in a myriad of studies and reviews of the
literature (22). Mercenier et al. list some potential health benefits attributed to lactic acid
bacteria which contains 39 distinct items ranging from specific immunomodulatory
properties such as an increase in immunoglobulin A (a gastrointestinal antibody)
production to a general effect of improving well-being (22). Despite the progress being
made identifying the health benefits of different probiotic strains, the knowledge of the
underlying mechanisms are not fully understood (18).
The process of selecting a probiotic strain begins with the identification of that
bacterium on the genus, species, and strain levels. If the bacterium does not possess
pathogenic characteristics then it should be further characterized at the technical,
physiological and functional levels. A potential probiotic strain of bacteria should meet
the following criteria in order to be a good candidate for use as a probiotic: the strain
must grow readily and be viable during transport and storage, the strain must be able to
resist the antimicrobial properties of the human gastrointestinal tract and adhere to
intestinal epithelial cells, the strain must also benefit the host either by competitively
excluding pathogenic bacteria or enhancing the growth of autochthonous microbes.
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Evaluating the Safety of Probiotic Bacteria
Regulatory authorities in Europe and North America aim to protect consumers
from pathogenic bacteria being marketed as probiotics. The European Food Safety
Authority (EFSA) recommends that new biologic products pass through a process by
which they receive a qualified presumption of safety (QPS) (18). This process involves
defining the taxonomic unit of the biologic product, assessing the body of knowledge on
the product, assessing its pathogenicity and evaluating its intended use. The American
counterpart to the EFSA is the Food and Drug Administration (FDA). The FDA regulates
probiotic agents as dietary supplements and so they place the burden of proof of safety
on the manufacturer (23). The FDA does not specify any criteria to prove safety, but
rather they note that scientific consensus must demonstrate safety or the manufacturer
must demonstrate safe historical usage of a probiotic agent. For the latter case, the
probiotic bacteria would be generally recognized as safe (GRAS) and would be
approved for sale in food products or as a supplement (18).
Guidelines for Evaluating the Safety of Bacterial Strains
The FAO/WHO working group report cites four possible types of side effects that
may occur from the consumption of bacteria marketed as probiotics. These side effects
were originally identified by Marteau in 2001 and serve as guidelines for evaluating the
safety of a bacterial strain before clinical trials (24). The side effects are:
Systemic Infections
Deleterious metabolic activities
Gene transfer
The excessive stimulation of the host’s immune response.
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Systemic infections
Documented occurrences of systemic infections from probiotic consumption are
few in number and have typically occurred in persons with existing medical conditions
(24). For example, four cases of Saccharomyces boulardii fungemia (fungus present in
host blood) were reported in hospitalized patients following S. boulardii therapy (25). S.
boulardii therapy is used to treat diarrhea in enterally fed and Clostridium difficile (a
pathogenic bacteria) infected patients (26).The investigators of this study cited that the
possible routes of infection could have occurred from patient or health provider contact
with intravenous catheters following the handling of S. boulardii packets. One of these
cases resulted in septic shock. The investigators recommended that S. boulardii
packets be handled outside of patient rooms and only with gloves to minimize their
inadvertent spread around susceptible patients (25). In two back to back ecologic
studies located in southern Finland, Saxelin et al. evaluated the prevalence of
bacteremia due to Lactobacilli consumption in 9229 probiotic consumers. This sample
population only produced 20 cases of bacteremia and no isolates of bacteria present in
the blood matched up with any probiotic strains being consumed (27). A follow up study
determined that the average incidence of bacteremia is 0.3 cases/100,000
inhabitants/year in Finland. Investigators concluded that probiotic use did not increase
the incidence of bacteremia (28).
Deleterious metabolic activity
Bacteria affect the metabolism of the small intestine via probiotic and dysbiotic
mechanisms. Some dysbiotic activities that negatively impact metabolic activity include
excessive deconjugation and dehydroxylation of bile salts and excessive degradation of
the intestinal epithelium’s mucosal layer (24). Listeria monocytogenes, a pathogenic
22
bacterium, deconjugate bile salts, this increases the bacteria’s growth rate and ability to
survive in the human gastrointestinal tract (27, 29). Deconjugation prevents the
precipitation of bile salts in the feces which results in the release of reactive nitrogen
species that accumulate in the colon and contribute to carcinogenesis (30).
Gene transfer
Gene transfer in bacteria occurs when a small molecule of DNA called a plasmid
is passed from one bacterial cell to another. Plasmids are a concern to scientists
because of their ability to spread pathogenic activity among bacteria. For example, if an
enterotoxin-producing strain of bacteria that possesses a plasmid is consumed, then it
is possible for that enterotoxin gene to be passed on to other gut microbes which would
otherwise be harmless (24). This is especially true for plasmids that transfer antibiotic
resistance, especially resistance to the antibiotic vancomycin. Vancomycin is used to
treat infections due to gram positive bacteria like C. difficile (31). These infections
typically occur after methicillin resistant staphylococci nosocomial infections and often
result in mortality because of the bacteria’s ability to survive antibiotic treatment (32).
Excessive immune stimulation
An excess of immunostimulation, can have deleterious effects on the overall
health of an individual (33). The body responds to physiological stressors like infection
or tissue injury through proinflammatory signals which are typically short lived (34). An
excess of this proinflammatory signaling can lead to chronic inflammation and disease
progression (34). Polysaccharides from the cell walls of bacteria typically elicit acute
responses from the host but the continued consumption of a probiotic that elicits this
response can lead to excess immune stimulation. Acute toxicity studies conducted in
murine models test for this excess immune stimulation and evaluate the effects of
23
consuming high single dosages of a bacteria. A probiotic should not cause dysbiosis
even when consumed at excessively high dosages (15, 35).
Helicobacter pylori is an example of a bacteria that induces excessive immune
stimulation (36). These bacteria invade human intestines and release reactive nitrogen
species which cause oxidative damage to intestinal epithelial cells’ DNA (36). Once the
DNA has been extensively damaged, the cells undergo apoptosis and are shed in the
feces. This elicits an immune response in an effort to rid the intestines of H. pylori.
Chronic H. pylori infection results in ulcers and is a risk factor for carcinogenesis due to
the accumulation of DNA damage in intestinal organs. Furthermore, chronic infection by
H. pylori acts as a compounding factor for further immunostimulation (37). This type of
excessive immunostimulation is one reason why probiotics should be non-invasive,
transitory colonizers of the human gastrointestinal tract.
In Vitro and Animal Studies
In vitro studies typically used to assess the safety of the probiotic bacteria include
genome sequencing of the strain for identification and characterization purposes,
evaluations of antibiotic resistance, gene transfer capability and its ability to withstand
environmental stressors such as gastric juices and stomach acid. Following the
completion of in vitro studies, animal studies are used to assess the pathogenicity,
acute toxicity, and gastrointestinal viability of the bacteria. Animals may also be used to
study probiotic mechanisms or actions that were observed during the in vitro testing
phase (16). If no adverse effects are observed in animal studies and the bacteria has
historically been used safely, then further testing can proceed to clinical trials.
24
Clinical Trials
Clinical trials involve human volunteers and are divided into Phases I, II, III and
IV (38). Phase I trials are conducted to determine the safety of the biologic agent and
typically consists of less than one hundred volunteers. These trials provide information
on possible symptoms associated with the biologic agent as well as determining a
maximally-tolerated dose (38). Phase II trials are typically conducted on diseased
populations and aim to provide information on the efficacy of the biologic agent to treat
a disease or condition. If improvement of the disease or health condition occurs, in
comparison with a placebo, then scientists will proceed to phase III trials. A phase III
trial expands on the knowledge gained from phase II trials and aims to determine the
advantage of the new biologic agent against standard therapy (38). Phase III trials also
provide information on drug interactions, side effects, and appropriate dosages.
Following the completion of a phase III trial for a new drug, manufacturers can request
to market the drug to the general public and upon approval of that request by a
regulatory agency, they will begin a phase IV trial (38). Phase IV trials are also known
as post-market surveillance studies because rare and slowly developing adverse effects
can more readily be monitored after the drug is made available to the public. Of
important note are recommendations made by the FAO/WHO when evaluating
probiotics in clinical trials. They recommend that during phase II trials (evaluation of
efficacy), validated quality of life assessment tools be used to determine the overall
impact of the probiotic on host health rather than only evaluating its effect on the
disease or health condition (16).
25
Quality of Life Assessment
Medical treatments for patients presenting with gastrointestinal diseases focus on
the treatment of the disease and at times fail to acknowledge the patients’ general well-
being (39). An evaluation of patients’ symptoms is necessary to assess not only the
reduction in severity of disease but also the overall effect of the intervention on patients’
lives. Gastrointestinal symptoms vary from person to person and often times these
symptoms lack a proper pathophysiologic identification. The subjective nature of
symptom intensity, however, makes it difficult to compare disease severity in different
patients. The development of questionnaire-based instruments to evaluate the presence
and severity of symptoms is necessary to enumerate and compare the overall effect of
conventional medicine across populations. The Gastrointestinal Symptom Rating Scale
(GSRS) is one such instrument. It was developed for the global rating of the severity of
symptoms based on the clinical interview and has been validated in six countries (40,
41).
Development of the GSRS
Svedlund et al. 1988 constructed the original Gastrointestinal Symptom Rating
scale in study participants who presented with irritable bowel syndrome and peptic ulcer
disease. The study evaluated the clinical efficacy of conventional medical therapy for
irritable bowel syndrome (n=101) and peptic ulcer disease (n=103) compared to an
intervention that utilized psychotherapy in conjunction with conventional medical
practices. The symptoms chosen for use in the questionnaire were based off of the
clinical experience of physicians and reports in the literature. The questionnaire
measured symptoms such as epigastric pain, heart burn, increased flatus and nausea;
26
a full list of symptoms is reported in Table 3-2. The original GSRS utilized four well
defined intensity scores ranging from 0 to 3:
0: No or transient pain
1:Occasional aches and pains interfering with some social activities
2: Prolonged and troublesome aches and pains causing requests for relief and interfering with many social activities
3: Severe or crippling pains with impact on all social activities
Undefined half steps were also used to measure symptoms at higher sensitivities (40):
Initial Validation of the GSRS
The validation of the GSRS was conducted using a subset of participants (n=20)
from the clinical efficacy study conducted by Svedlund et al. The participants were
administered the GSRS in an interview format. Medical records from the past month in
addition to patient responses were utilized to rate the participants with the GSRS. Each
study participant was interviewed and rated twice by two psychiatrists in order to
eliminate interviewer bias. The inter-rater agreement of GSRS scores, determined via
the interview process, was tested for using Cohen’s weighted kappa (Kw) which ranges
in score from 0 to 1. The maximum score of 1 indicates a perfect agreement between
interviewers while a score of 0 indicates no agreement between interviewers (40). A
higher Kw score indicates that the GSRS can be used to accurately gauge the intensity
of the gastrointestinal symptoms it inquires about. All weighted kappa scores measured
were high (ranging 0.86 to 1.00 for individual symptoms and 0.92 to 0.94 for
syndromes) indicating inter-rater agreement was not likely due to chance (40). Despite
high inter-rater agreement, the original GSRS was only utilized to detect the presence
or absence of symptoms. Svedlund et al. did not test the ability of the questionnaire to
27
detect changes in gastrointestinal symptoms. This severely limits its use in clinical trials
as a diagnostic instrument. Furthermore, Svedlund et al. used an interview process to
administer the questionnaire. In clinical trials, the interview method is unrealistic due to
increased costs and burden on study participants. The investigators concluded that
more studies were necessary in order for the GSRS to be suitable as a diagnostic
instrument in clinical trials.
An Updated GSRS
Revicki et al. 1998 updated the original translations of the GSRS (Svedlund et al.
1988) with simpler terminology replacing words like borborygmus and eructation with
stomach rumbling and burping (42). These changes made the GSRS easier to
understand and increased its appeal for use as a self-administered questionnaire. The
GSRS was validated with respect to the Psychological and General Well-Being scale
(PGWB) and the Medical Outcomes Study short form (SF-36) which evaluate quality of
life. Revicki et al. conducted the validation study using participants (n=516) of a clinical
trial which evaluated Ranitidine as a treatment for gastroesphogeal reflux disease
(GERD). Previous clinical trials utilizing the PGWB and SF-36 in GERD patients
demonstrated the responsiveness of those questionnaires to experimental treatment
(43) (44). Revicki et al. reasoned that if the results generated by the GSRS correlated
well with the PGWB and SF-36 then they could be used together as holistic measures
of experimental treatments rather than only evaluating a reduction in symptoms (39).
The validation process involved evaluating the GSRS’s internal consistency reliability,
construct validity, discriminant validity and responsiveness (42).
28
Internal consistency reliability
The internal consistency reliability of a questionnaire is a measure of whether or
not the questionnaire means the same thing to different people. Due to the subjective
nature of gastrointestinal symptoms it is necessary for the GSRS to evaluate symptoms
using overarching constructs (42). For example, the GSRS contains a constipation
syndrome which is a mean of three different symptom scores: constipation, feelings of
incomplete evacuation and hard stools. These three symptoms evaluate an overarching
construct, constipation, by asking about some characteristics that are clinically
associated with constipation.
The Cronbach α test statistic is used to measure internal consistency reliability
and ranged from (0.61 to 0.80) for the various syndromes of the GSRS (42). A higher
Cronbach α score (maximum 1, minimum 0) indicates that the symptoms are very
similar in relation to the overarching construct. The Cronbach α score of the constipation
syndrome was 0.80, this is at the upper bound of “good” internal consistency reliability
and indicates that the symptoms are an adequate measure of the syndrome (42).
Next, Revicki et al. used intraclass correlations coefficients (ICC) to measure
how alike the syndromes were between study participants. Higher ICC (maximum 1,
minimum 0) scores indicate that the syndromes meant the same things to different
participants. ICC scores ranged from 0.42 to 0.60. These scores indicate fair to good
agreement between participant’s when taking the self-administered GSRS (42). While
these ICC scores may appear low they provide valuable insight into the validity of the
questionnaire. Symptoms are subjective by nature and so they should inherently mean
different things to different people. An ICC score that is too high would indicate that the
questionnaire does not adequately measure variability and a score that is too low
29
indicates that the questionnaire allows too much variability in its possible answers. For
example, if a questionnaire asked participants to answer the question, “How old are
you?” the expected ICC score would be very low due to the high variability of possible
answers. In contrast, a perfect score of 1 would indicate that all participants answered
exactly the same to the question. The fact that the internal consistency reliability and
intraclass correlation coefficients of the GSRS falls into the fair to good score range
indicates that it is a true measure of subjective gastrointestinal symptoms. The GSRS is
both sensitive enough to detect differences between study participants and similar
enough to mean the same thing to them (42).
Construct validity
The construct validity of the GSRS is a measure of how well it correlated with the
PGWB and SF-36 quality of life questionnaires. Pearson product moment correlations
were utilized to measure the GSRS’s construct validity (42). Revicki et al., in agreement
with Dimenas et al. and Svedlund et al., hypothesized that GSRS syndrome scores
would negatively correlate with PGWB and SF-36 subscales (Table 1-1). This
hypothesis is based on the idea that gastrointestinal symptoms adversely affect the
general wellness and psychological wellness of individuals (39, 40).
Pearson correlations of the GSRS syndromes and SF-36 sub-scales were all
negative and ranged from -0.12 to -0.44. The reflux syndrome was the only GSRS
syndrome that failed to significantly correlate with the SF-36 in five sub-scales (physical
and emotional role limitations, mental health, and mental component summary and
physical component summary. While the correlations were negative for these five sub-
scales they were not significant (p>0.05). Pearson correlations of the GSRS syndromes
and PGWB sub-scales were all negative and ranged from -0.09 to -0.41. A majority of
30
the GSRS syndrome and PGWB sub-scale correlations were significant (p<0.0001).
The positive well-being subscale did not significantly correlate with any GSRS
syndrome and the reflux syndrome failed to significantly correlate with depression,
positive well-being and behavioral control PGWB subscales. Revicki et al. conclude that
the GSRS has an acceptable construct validity but they fail to discuss the non-
significant Pearson correlations. Revicki et al. never describe which GSRS values
(baseline or treatment) were used to calculate the Pearson correlations. Considering
that the reflux syndrome correlations were consistently not significant, in a study
population that was receiving Ranitidine therapy to treat GERD, it is likely that the
investigators used treatment GSRS scores to calculate Pearson correlations. This
diminishes the quality of their results because correlation scores could be
underestimated due to Ranitidine’s reduction in symptom severity. For example, the net
change of the reflux syndrome score was -1.23 after 6 weeks of treatment indicating
that PGWB and SF-36 scores would be higher if GSRS scores during the treatment
period would be used. This would reduce the correlation score thus underestimating the
predictive potential of the GSRS syndromes on quality of life. Overall Revicki et al.
conclude that the construct validity of the GSRS is consistent with previous findings (39)
and they support its continued use as an indirect measure of quality of life.
Discriminant validity
Revicki et al. also examined the discriminant validity of the questionnaire which
evaluates the effectiveness of the questionnaire to identify treatment responders from
treatment non-responders (42). Participants in the study were randomized to Ranitidine
intervention or Omeprazole (standard therapy) for the treatment of gastroesphogeal
reflux disease. They were administered a GSRS once at baseline and once after 6
31
weeks of intervention (42). Mean GSRS syndrome scores of treatment responders and
treatment non-responders were compared with a t-test. All syndrome scores were
statistically different between treatment responders and non-responders after 6 weeks
of Ranitidine intervention. The largest change occurred in the reflux syndrome (net
change of -1.23 compared to baseline) as expected. The GSRS was sensitive enough
to detect a net change in syndrome scores as low as -0.25 indicating it is a useful tool in
the evaluation of gastrointestinal symptoms of GERD patients. To further evaluate the
discriminant validity of the questionnaire, Revicki et al. compared physician-assessed
symptom ratings to self-reported ratings from the GSRS. It was shown that the
questionnaire was able to rank symptom severity in a predictable manner when
compared to that of the physician assessed ratings. When GERD patients reported 2 or
3 symptoms to their physicians the GSRS syndrome scores scaled accordingly. This
data demonstrates the ability of the GSRS to detect changes in severity of symptoms
akin to physician-based interviews.
Responsiveness
Responsiveness of participants to the GSRS was measured by evaluating the
net change of treatment responders’ and non-responders’ mean GSRS scores (42).
Responders to Ranitidine therapy experienced a net decrease in mean reflux syndrome
scores of 1.23 compared to a decrease of 0.46 using standard therapy (p<0.0001). This
indicates that the GSRS is sensitive enough to detect changes in reflux syndrome
scores in response to differing GERD treatments. The conclusion Revicki et al. drew
was that the responsiveness measured can be used in populations presenting with
other gastrointestinal disorders as well as those with GERD, though they acknowledge
32
that more validation studies should be conducted to ensure its responsiveness in those
populations (42).
The GSRS is an easy to understand self-administered questionnaire that
evaluates a variety of gastrointestinal symptoms. This questionnaire has to date been
validated and used in a number of gastrointestinal disorders including irritable bowel
syndrome, gastric ulcers, and GERD. However, its use has not extended beyond sick
populations. Traditionally, symptom questionnaires are useful for detecting a reduction
in symptom severity or duration and so their use in clinical trials is encouraged, but
gastrointestinal symptoms are not exclusively experienced in sick or diseased
populations. Healthy individuals can also experience gastrointestinal symptoms typically
due to changes in diet and so, if the GSRS is able to detect decreases in syndrome
scores, it should, in practice, be able to detect increases in syndrome scores in healthy
populations. However, to date, no studies have evaluated the validity of the GSRS in
healthy populations.
Determination of Healthy Status in Research Participants
The composition of a study’s participants has an impact on the interpretation of a
study’s findings. For example, a study that evaluates the efficacy of a new cancer
treatment would be meaningless if the new treatment was not tested in cancer patients.
This example demonstrates the necessity of having a well-defined population during a
clinical trial. When study populations are chosen well, the study’s conclusions have a
higher degree of validity because results are not being affected by external variables.
External variables that can alter data are called confounders or confounding variables
(38). Scientists control for confounding variables through study design. In clinical trials,
study participants are usually the largest source of confounding variables simply due to
33
the fact no human is physically or socially identical to another. A method by which study
participants can be controlled for is by defining inclusion and exclusion criteria for study
volunteers. Inclusion and exclusion criteria in phase I clinical trials evaluating probiotics
aim to prevent the active enrollment of individuals with preexisting gastrointestinal
disorders. Investigators are able to ensure that any gastrointestinal symptoms
experienced during the study are not a result of probiotic consumption by excluding
volunteers with gastrointestinal disorders from taking part in the study. Another method
used to prevent the introduction of confounders is through the use of questionnaires.
Questionnaires serve as both screening and diagnostic tools depending on the context
of their use. One such questionnaire is the Global Physical Activity Questionnaire
(GPAQ).
In a systematic review of 89 studies involving physical activity questionnaires,
Helmerhost et al. tested the reliability and validity of the GPAQ using intraclass
correlation coefficients (ICC), Pearson and Spearman correlation coefficients and
Cohen’s weighted kappa. Pearson and Spearman correlation coefficients and Cohen’s
weighted kappa are described in the GSRS section (45). The intraclass correlation
coefficients, much like Cohen’s weighted kappa, were used to measure the reliability of
a quantitative measure made by two different interviewers when a questionnaire was
not self-reported.
The Global Physical Activity Questionnaire was developed by the World Health
Organization in 2002 to identify inadequate physical activity levels, a key risk factor for
disease. The questionnaire classifies an individual’s activity level based on a
combination of its intensity (classified as vigorous, moderate, and light) and duration
34
(measured in minutes/week) in a typical week. Bull et al. are the main contributors to the
validation of the GPAQ and they conducted a study where over 3600 participants were
either administered or received a self-report version of the GPAQ on their first
consultation, at this time they were given a pedometer or accelerometer in order to
provide an unbiased assessment of their physical activity level. On their second and
third clinical visits, they were given the questionnaires again in order to assess reliability
and validity. Construct validity was measured by comparison with an instrument
developed by the investigators based off the International Physical Activity
Questionnaire, which has previously been validated (46). The International Physical
Activity Questionnaire is administered in a short and long form, both of which have their
own strengths and weaknesses. The short version lacks domain specific estimates and
the long version contains 27 items which is too cumbersome for administration in large
clinical trials. The GPAQ is an attempt at capitalizing on the strengths of the short (easy
to use and administer) and long (accurate estimates of duration and intensity) versions
of the international physical activity questionnaire.
The GPAQ measures physical activity levels under three domains: leisure and
recreation, work, and transportation. Activity level is defined as either moderate or
vigorous and is qualified by frequency in the past week. Bull et al. reported that
reliability coefficients presented with moderate to substantial strength, construct validity
between GPAQ and the IPAQ instruments also showed moderate to strong positive
correlations but criterion validity was only in the poor to fair range. These results are
overall very positive as the GPAQ has demonstrated consistent activity level scoring
across several different cultural regions such as Ethiopia, Indonesia, India and Japan.
35
Criterion validity, which objectively measures physical activity with pedometers or
accelerometers provided to participants, resulted in poor to fair agreement with self-
reported values. The investigators explained this by stating that the GPAQ should be
evaluated in more countries to decrease the influence of a particular culture or
geographic region on the data collected. The issue that Bull et al. faced with using
pedometers and accelerometers is that they only measure movement of the body and
may not accurately represent how much physical exertion a person experiences. This
diminishes the value of comparing pedometer measurements against self-reported
activity levels. Overall, the use of the GPAQ as a consistent and reliable measure of
self-reported activity level does not have the best consistency and validity. Study
participants can unknowingly over or under estimate exercise levels due to the
subjective nature of physical exertion. A study participant who only recently began
cardiovascular exercises may consider walking at a brisk pace vigorous while a
seasoned marathon runner would only consider it as light in intensity. This subjective
variability in self-reported physical activity levels can introduce recall bias into data
collected. With this in mind, the GPAQ is likely not the best tool for evaluating the
efficacy of physical activity on health conditions because more reliable and unbiased
tools, such as daily diaries, can be used for this purpose. These limitations lead to the
conclusion that the GPAQ is better suited for evaluating the perceived activity level of a
given sample and not as a diagnostic measure. This makes it an ideal screening tool.
Bacillus subtilis as a Probiotic
Bacillus subtilis is a gram positive, spore forming, bacteria that is most commonly
found in the soil (47). Bacillus spores are unique because of their resistance to stomach
acid and pancreatic juices, which increases their chances of survival through the human
36
gastrointestinal tract. It is hypothesized that once the spores have reached the large
intestine they begin to germinate where they can exert probiotic effects (48). Bacillus
subtilis R0179 is currently marketed throughout Asia as Medilac-DS/S®, S/DS refer to
single and double strength respectively, in a 1:9 formulation with Enterococcus faecium
R0026 (DS dosage: 6x109 CFU/d = 2 caps). This probiotic formulation has been
evaluated in approximately 100 clinical investigations and evaluated by two systematic
reviews (49). The studies, reviewed by Tompkins et al. 2010, evaluated the efficacy of
the probiotic in several different disease states. A majority of these studies were
published in the Chinese, Japanese and Korean language journals making it difficult to
directly evaluate each study. Tompkins et al. 2010 also had limited access to some
studies forcing them to rely solely on English abstracts for their review.
Tompkins et al. 2010 first report on the ability of Medilac-S® to change the
microbiota composition of cirrhotic liver patients to resemble that of healthy controls.
Fifty cirrhotic liver patients and 20 healthy volunteers were randomized to Medilac-S® or
Bifico (a probiotic containing Bifidobacterium sp.-undisclosed, Lactobacillus acidophilus
and Enterococcus sp.-undisclosed in a total dosage of 3x109 cfu/day). Intestinal
microbiota analysis revealed a significant difference at baseline that was resolved after
two weeks of intervention (2 capsules at 3x109 CFU/capsule). While this data provides
some evidence towards the impact of B. subtilis R0179 on the intestinal microbiome,
Tompkins et al. 2010 caution classifying this shift in intestinal microbiota as a health
benefit. Furthermore no data was collected evaluating the long term effects of this shift
in intestinal microbiota.
37
Second, Tomkins et al. 2010 report on the evaluation of Medilac-DS® use, in
conjunction with traditional therapy, to eradicate Helicobacter pylori in gastric ulcer
patients (n=352). Probiotic intervention lasted for 8 weeks. The study resulted in an
83.5% H. pylori eradication rate in the probiotic group compared to 73.3% in the control
(p=0.027). Patients randomized to the probiotic intervention also experienced less
diarrhea and fewer overall symptoms compared to traditional therapy. The investigators
believe that the decrease in total side effects resulted in an increase in drug compliance
which in turn increased eradication rates. These results are promising for the continued
use of Medilac-DS as adjuvant therapy for H. pylori eradication. The reduction of
symptoms, however, is not entirely unique to Medilac-DS. Several other studies have
demonstrated that probiotic formulations composed of Lactobacilli, Bifidobacterium and
fermented milk products like Kefir, also reduce symptoms associated with traditional H.
pylori eradication (50-52). A major limitation of these studies is their use of generic
questionnaires which have not been validated in H. pylori patients. This immediately
casts some doubt on their symptom data and its ability to adequately detect decreases
in symptom intensity and presence. The investigators caution that while there was an
increased eradication rate of H. pylori while consuming Medilac-DS® this may only be
due to an increase in compliance and not a direct result of interaction between E.
faecium, B. subtilis, and H. pylori.
Third, Tompkins et al. 2010 report on four double-blind, placebo-controlled trials
that evaluated the effect of probiotic interventions, in addition to traditional therapy, on
irritable bowel syndrome. These clinical trials resulted in significant decreases in
abdominal pain, but no reduction in gas expulsion, bloating, defecation or other
38
gastrointestinal symptoms commonly reported in irritable bowel syndrome patients. The
most promising of these studies was a randomized, double blind, placebo-controlled
clinical trial (n=34) which utilized Medilac-DS® (4 weeks) as mono-therapy for IBS. This
study resulted in a significant reduction in frequency (p=0.038) and severity of
abdominal pain as measured by a visual analog scale (p=0.044). The main text of the
article is published in Korean and so access to details of study design is limited.
Tompkins et al. 2010 do not report on whether frequency and severity were measured
over the entire 4-week intervention period or only in the baseline and post intervention
time point. If the reduction in frequency and severity of abdominal pain occurred only in
week 4 of intervention and not in weeks 2 or 3, then much doubt is cast on the ability of
Medilac-DS® to reduce IBS symptoms.
Fourth, Tompkins et al. 2010 report on seven clinical trials (n=745) evaluating
Medilac-DS/S® use in participants with acute, chronic, and antibiotic-associated
diarrhea. The studies yielded some positive results but Tompkins et al. could find no
reason to cite the Medilac-DS/S® probiotic interventions as the cause partially due to
study design. Only one of the studies evaluating the various diarrhea types was blinded.
This study compared the effects of Medilac S® and Pei Fei Kang® (undefined probiotic)
on acute and chronic diarrhea in a randomized, single-blind, placebo-controlled trial
(acute diarrhea: n=95, chronic diarrhea: n=48). The intervention period was 5 days and
there was no difference detected between groups. It is unclear whether the primary
outcome of this study was to treat diarrhea or to compare the effect of the different
probiotics. This study could have benefitted from a placebo arm to evaluate overall
efficacy of the probiotics. While the study was single blinded, the participants would
39
have still been notified that they would be randomized to a probiotic, either Medilac S®
and Pei Fei Kang®, thus diminishing the benefits of blinding. Any results demonstrating
a benefit attributed to the probiotics may be confounded due to the placebo effect.
Fifth, Tompkins et al.2010 report on ten clinical trials (n=528) evaluating the
effect of Medilac-S® in ulcerative colitis patients. These studies were randomized and
used active controls (traditional therapy) but were not blinded. The studies
demonstrated a 12%-31% increase in the treatment of ulcerative colitis when traditional
therapy was combined with the consumption of Medilac-S (3x109 CFU/day).
Intervention periods ranged from 2-12 weeks. Tompkins et al. 2010 concluded that
these studies were adequate in terms of participant selection, randomization and dosing
regimens but that they were poorly designed in terms of blinding, assessment of
compliance and statistical analyses which casts doubt on their results.
Of the studies reviewed by Tomkins et al. 2010 that reported adverse events no
serious adverse events or reactions could be linked directly to the probiotic. No cases of
bacteremia or septicemia occurred. A handful of symptoms were reported among the
various studies including nausea, vomiting, dizziness and feelings of being flustered, but
these symptoms did not persist and were isolated to only a handful of individuals that
were randomized to the probiotic interventions. Despite the formidable research on this
probiotic formulation, there are no studies which evaluate the B. subtilis R0179 strain as
a single probiotic agent (49).
Tompkins et al. 2010 commented on the validity of some conclusions drawn from
the reviewed studies. They cite poor study designs (49). Tompkins et al. 2010 also
conclude that randomized, double-blind, placebo-controlled clinical trials are necessary
40
before any symptom reduction or increased efficacy can be attributed to probiotic
therapy with a high degree of confidence (49). More work must also be done on the
analysis of the probiotic based on dosage levels which would, in turn, necessitate closer
monitoring and better reporting of symptoms and adverse events in the populations
being studied.
The continued acceptance and usage of probiotics in different regions of the
world has an effect on the popularity and acceptance of these products for their
therapeutic properties. Both patients and their physicians will increasingly find
themselves in situations where they need to understand whether a particular type of
probiotic supplement can have a beneficial effect on a disease state. Due to the
increasing costs of health care and the monetary and symptomatic burdens some drugs
place on patients, probiotics are continuously being turned to as alternative and
supplemental health care. It is with these reasons in mind, along with the general
understanding of safety of a particular strain, that more research needs to be
conducted. Tompkins et al. 2010 have presented some evidence that B. subtilis R0179
is a safe product when consumed with E. faecium R0026, but its use in diseased
populations must be examined more closely (47). This warrants the use of randomized,
double-blind, placebo-controlled studies that utilize validated health-related quality of life
questionnaires and microbial sequencing techniques to assess the viability of the
bacteria post ingestion. When a health benefit is suspected, molecular techniques
identifying relevant biomarkers should exist to substantiate any related improvement in
quality of life as determined by questionnaires.
41
In Vitro Studies
The Safety of Bacillus subtilis
The usage of the spore forming Bacillus genus of bacteria as a probiotic has
grown in popularity. A study by Sorokulova et al. conducted on Bacillus subtilis BS3,
taken from the Ukrainian Collection of Microorganisms, cultured the strain to evaluate its
safety. 16S rRNA sequencing was conducted to identify the bacteria via an identification
key. Cultured bacteria were also evaluated for antibiotic resistance, the presence of
plasmids, enterotoxin genes and virulence factor analysis including enterotoxin
detection. Adhesion toxicity studies were carried out concurrently in a murine model
(n=220) (53).
Strain BS3 was identified as a gram positive, rod shaped bacteria capable of
forming endospores with the ability to synthesize catalase, an enzyme that catalyzes
the degradation of hydrogen peroxide to water (53). Hydrogen peroxide acts as an anti-
microbial in the human gastrointestinal tract and is classified as a reactive oxygen
species. Bacteria that can neutralize reactive oxygen species have a greater chance of
surviving mammalian gastrointestinal tracts, though this trait is not desirable in
pathogenic bacteria (54). Strain BS3 lacked a plasmid and was susceptible to all
antibiotics mandated by EFSA (53) . BS3 was resistant to Oxacillin and was moderately
resistant to Amoxicillin, Methicillin and some Cephalosporins. This was not a concern to
Sorokulova et al. as Bacillus subtilis is non-pathogenic and the resistance was below
the maximally-recommended resistance concentrations as determined by regulatory
bodies. BS3 presented low adhesion rates to mucin and Caco-2 cells, no enterotoxin
genes were found after DNA analysis of the bacteria and no virulence factors (such as
the Hbl gene which is often associated with diarrhea induction) were found (53). The in
42
vivo acute toxicity study subjected mice (n=220) to high dosages of BS3 orally (5x107 to
2x1011 CFU/mouse) and intravenously (5x107 to 5x109 CFU/mouse). No treatment-
related deaths or declines in general health (decreased activity and weight gain)
occurred. No mice exhibited chronic toxicity symptoms (53). Although the results of this
study cannot be translated to all strains of B. subtilis, they are good examples of how
the safety of a particular bacterial strain should be evaluated before clinical trials.
Sorokulova et al. also evaluated the chronic toxicity of other B. subtilis strains in
mice, guinea pigs and rabbits (53). Ten New Zealand White rabbits were exposed to
daily oral dosages (1x109 CFU/day) of the spore form of B. subtilis for 30 days. Animals
were then sacrificed and blood was drawn and tissue samples were taken of the
visceral organs for histology. The rabbits showed no adverse symptoms or side effects
from consumption of the bacteria. Histology of the organs and lymph nodes showed no
change in bacterial content indicating no bacteremia (53). The chronic toxicity study was
also done on guinea pigs where a single dosage of spores was given at 1x109 CFU/day.
After 17 days of monitoring the, generally sensitive guinea pigs, showed no
abnormalities in appetite, behavior, feces and weight (53). No toxicity levels were
observed in either control or treatment groups.
The Safety of Bacillus subtilis R0179
The use of B. subtilis R0179 in the probiotic formulation Medilac DS/S as
described previously carries a considerable amount of evidence towards its safety for
human consumption. In an effort to further evaluate the safety of this particular strain,
Tompkins et al. 2008 conducted genetic identification, plasmid analysis, enterotoxin
analysis, antibiotic resistance, human intestinal epithelial adhesion, and acute toxicity
studies (47).
43
The genetic identification of strain R0179 confirmed its inclusion in the Bacillus
subtilis genus and species (47). This test was conducted with 16srRNA sequencing with
a 100% match rate to a standardized B. subtilis genome sequence. Strain R0179 lacked
plasmids and DNA analysis revealed the absence of the toxin genes hbl, nhe and cytK
when compared to Bacillus cereus (a positive control). These enterotoxins genes are
linked to the production of enterotoxins and have been implicated as the cause of B.
cereus’ classification as a food borne pathogen (55). Antibiotic resistance analysis
revealed that strain R0179 was below maximally-recommended resistance levels for all
antibiotics tested (47). Adhesion tests revealed that strain R0179 minimally adheres to
human intestinal epithelium cell models. Its adhesion rates are about 5 times lower than
those of Lactobacillus helveticus, a bacteria commonly used in fermented milk products
(56).The acute toxicity study, a 28 day study in which 5 female pathogen free rats were
fed R0179 spores (2x109 CFU/kg/day), showed no signs of toxicity, oral intolerance,
loss in body mass, decreased food consumption, or increased mortality (47).
Additionally organ mass to body mass ratio were not significantly different from control
and strain R0179 did not migrate into the rats’ livers or spleens indicating no bacteremia
or septicemia. Expectedly strain R0179 was found at high levels in the intestinal
contents of the rats (1.5x106-1.2x107 CFU/g).
Animal Studies
Germination of Bacillus subtilis in the Gastrointestinal Tract
Bacillus subtilis’ spore form confers upon it many beneficial factors like high
resistance to wet and dry heat, ultra-violet and gamma radiation, desiccation and
oxidation (57). B. subtilis is most often consumed while it is in the spore form which
provides it resistance to the acidic contents of the stomach (58). The bacteria, as a
44
whole, are largely aerobic though they can be induced into an anaerobic state. This
limits the viability of the bacteria in the anaerobic environment of the intestinal tract, and
so, before any probiotic effect can be attributed to B. subtilis, the mechanism of its
passage and survival in the GIT must be understood (48).
In an effort to discern the fate of ingested Bacillus spores Spinosa et al. isolated
B. subtilis MO1099 and inoculated 9 BALB/c mice at a dose of 109 spores. The mice
were then starved for 16 hours and sacrificed at different times post inoculation (58).
Two mice were inoculated in the gastrointestinal tract with the spore form of the
bacteria. The spores were found in all portions of the intestines, but diminished over a
period of three days whereupon they reached levels below the detectable limit. Spinosa
et al. concluded that the bacteria did not germinate into vegetative cells in the ileum,
jejunum or in the feces. The investigators concluded that any probiotic effect from B.
subtilis may in fact be due to the spore form of the bacteria (58). Their acute toxicity
study results are in line with the studies conducted on other B.subtilis strains by
Sorokulova et al. (53) and Tompkins et al. (47).
A study conducted by Casula et al. evaluated how spores of Bacillus, inoculated
(2x108 CFU/ml given in a 0.2 ml suspension) in one-week-old BALB/c mice (n=6),
germinated in the gastrointestinal tract (48). The bacteria were genetically modified to
strongly express an engineered ftsH-lacZ gene while in the vegetative state. The ftsH
gene codes for an ATPase (59) while the lacZ gene, commonly referred to as a reporter
gene, is derived from Escherichia coli genetic material and is useful for detection assays
(56). When the mice were sacrificed, an RNA primer for this genetically-engineered
gene was used to run rt-PCR allowing the investigators to detect spore germination in
45
the mouse gastrointestinal tract. This novel method, originally developed by Lysenko et
al. 1997, was supplemented by the collection of mice feces in order to evaluate the
transit viability of the inoculated Bacillus spores. The investigators were able to detect
as low as 102 vegetative cells, in the mouse intestinal tract. The vegetative bacteria
were found primarily in the jejunum of the mice and at levels that were only lower than
the inoculation concentration by a factor of ten. Investigators concluded that due to the
acidic composition of duodenal juices, germination may be inhibited in that portion of the
small intestine (48). While there may have been some germination in the duodenum,
the sensitivity of the assay used to detect the engineered gene may be to blame for the
inability to detect the vegetative cells in that portion of the intestines.
Casula et al. also observed that the highest concentration of Bacillus spores
were found 18 to 24 hours post inoculation indicating that both germination and spore
formation is occurring concurrently (48). This information, paired along with the fact that
vegetative cells were found post inoculation, led them to conclude that while Bacillus
may begin to colonize in the gastrointestinal tract some environmental factor prevents
its permanent colonization and thus it must resporulate leading to its subsequent
excretion in the feces. This may be due to its low adhesion rates as demonstrated, in
vitro, by Tompkins et al. 2008 on a model of human intestinal epithelial cells and
Sorokulova et al. in mucin and Caco-2 cell lines (47, 53). Any probiotic effect exerted
would likely occur during this brief colonization period wherein the bacteria could act
either as a competitive exclusion agent by briefly adhering to the intestinal epithelium
and thus prevent the adhesion of pathogenic bacteria or by some other probiotic
mechanism (48).
46
While the studies by Casula et al. and Spinosa et al. are contradictory with
regards to the ability of Bacillus spores to germinate in the gastrointestinal tract of mice,
it is important to consider the advantages of the rt-PCR assay used by Casula et al.
(48). This technique was able to detect levels of the vegetative cells far below those
typically observed with the common plating technique used by Spinosa et al. (58).
Furthermore, both studies contribute evidence towards the conclusion that B. subtilis
temporarily inhabits the gastrointestinal tract of mammals.
B. subtilis in the Human Intestinal Tract
Bacillus species have traditionally been considered inhabitants of soil due to their
spore forming capabilities. Research has shown, however, that once Bacillus spores
enter the gastrointestinal tract of mice, they can enter the vegetative state and
resporulate (48). However, this had not been previously demonstrated in humans (48).
Hong et al. hypothesize that Bacillus species may be permanent residents of the human
gastrointestinal tract and are only more commonly found in soil due to their spore
forming capabilities (60). The theory is that once Bacillus spores germinate in the small
intestine of man and animal they begin to outgrow their environment. As the vegetative
cells progress into the colon they resporulate where they are excreted along with feces.
This leads to their accumulation in soils. This theory is supported by evidence of
Bacillus spores being recovered from human feces (61) as well as their historical
identification as soil organisms (62).
To further elucidate the nature of Bacillus subtilis residence in the human
gastrointestinal tract Hong et al. conducted a study in healthy human volunteers. They
first retrieved fecal samples from study participants (n=29) who had not previously taken
any probiotic within the past year. Next, they retrieved two biopsies from the proximal
47
ileum of six ileostomy patients (60). The ileostomy patients did not have a history of
antibiotic, probiotic or laxative use at least one month before surgery. Hong et al.
reported that Bacillus spores were identified in the fecal samples collected from both
sets of participants (60). They estimated that human feces may carry on average 104
spores per gram of feces while the small intestine may carry 109 spores per gram or
greater. Hong et al. acknowledge that they did not control for extraneous sources of
Bacillus species found in food and so this could contribute to inflated estimates.
After isolating B. subtilis from the fecal and ileostomy samples, Hong et al.
proceeded to identify some key characteristics of the bacteria which may contribute to
their ability to live as gut commensals. The isolates were confirmed to grow and
sporulate under anaerobic conditions which is useful for the bacteria as they move into
the large intestine (60). It was also observed that the B. subtilis isolates were able to
form biofilms which serve to protect vegetative cells from environmental stressors like
antimicrobials and gastric juices. The biofilm has also been shown to stimulate
repopulation of the vegetative cell (63). Hong et al. conclude that a diverse community
of Bacillus species reside in the human gastrointestinal tract and that B. subtilis is likely
a gut commensal, but their total impact on the intestinal microbiome remains a mystery
(60).
Potential Health Benefits of Bacillus subtilis
The Effect of B. subtilis on Immune Function
A popular strain of the Bacillus subtilis is used in Japan for the fermentation of
cooked soybeans (called Natto) and has been shown to have probiotic properties (58).
This process of fermentation has resulted in the unique naming of Bacillus subtilis as B.
natto or B. subtilis var. natto (58). The effect of B. natto on the B and T lymphocytes in
48
the spleens of chickens was investigated in 1986 by Inooka et al. Chicks were fed B.
natto supplemented feed (107 cfu/day) for 27 days and then sacrificed. Analysis of T
and B lymphocytes in the spleens showed that chicks fed the supplemented feed had
more T and B lymphocytes compared to those on a normal diet. This suggests that the
B. natto strain has some effect on immune functions in chicks (64).
Popular probiotics which have well established probiotic effects, such as
immunostimulatory properties, are Lactobacillus species (65). An in vitro study,
conducted by Hosoi et al. investigated the effect of B. subtilis natto on the viability of
human derived Lactobacillus reuteri and Lactobacillus acidophilus strains that were
independently co-cultured with B. subtilis in an aerobic environment (66). Hosoi et al.
observed an increase of viable cells of both L. reuteuri (4 log increase) but not in L.
acidophilus (1 log decrease) when co-cultured with B. subtilis for 6 days when
compared to independently cultured Lactobacilli spp. This positive relationship extended
beyond enhanced growth. When toxic levels of hydrogen peroxide were added to the
cultures, B. subtilis natto was able to prolong the life of L. reuteri by three days whereas
L. acidophilus cells perished much faster than independently co-cultured samples. This
increased viability was hypothesized to be a result of the enzyme subtilisin, produced by
B. subtilis which catalyze the degradation of hydrogen peroxide to water similar to many
other catalases. The study shows promise due to its intention to link growth promoting
properties B. subtilis (natto) with that of other probiotics. Of important note is the fact
that this particular B. subtilis (natto) strain is used in the manufacture of food products in
Japan (66). Alongside this is the fact that the investigators used human derived
Lactobacilli strains which indicates that the effect may also be observed in vivo (66).
49
Although this study provides evidence towards the conclusion that B. subtilis has a
positive impact on the viability of certain Lactobacilli strains it does not acknowledge
that these benefits may not impact the human intestinal microbiome on a biologically
significant scale.
The Effect of B. subtilis on Bone Metabolism
In previous studies, Tsukamoto et al. established that B. subtilis (natto) had a
positive impact on the production of Vitamin K2, a vitamin has been shown to take part
in the prevention of bone loss particularly in older populations suffering from a decline in
bone density (67). Vitamin K2 plays a role in the carboxylation of osteocalcin which is an
osteoblast protein used in the formation of bone matrix and is found abundantly in natto
a food commonly eaten in Japan. When the natto has been fermented with B. subtilis
vitamin K2 levels increase from 775µg/100g of natto to 1298µg/100g of natto (67). To
evaluate the effect of natto Tsukamoto et al. conducted a study (n=134) to evaluate the
effects of natto consumption on serum vitamin K2 and osteocalcin levels. This study was
not a direct consumption of B. subtilis, but rather an example of how B. subtilis can be
utilized as a functional component in food production.
Results of the study demonstrated that people who fell into a category of
occasionally (a few times a month) or frequently (a few times a week) consuming natto,
supplemented with B. subtilis, had significantly higher levels of serum vitamin K2
compared to those that only consumed natto rarely (less than a few times a month)
(67). This is coupled with evidence of higher serum levels of osteocalcin present in the
occasional and frequent groups indicating higher bone repairing activity. People who
frequently ate natto had vitamin K2 levels increase by us much as 2.5 ng/ml, while
osteocalcin only increased in men by as much as approximately 3 ng/ml. This study
50
demonstrates great promise as a method by which B. subtilis can be utilized as a
probiotic agent.
The Use of B. subtilis as Bowel Preparation for Colonoscopy
Colonoscopy viewing is a medical procedure used to evaluate the presence of
colonic lesions in humans which can develop into colon cancer. To prepare for the
procedure, patients are asked to consume either polyethylene glycol or sodium
phosphate solutions (68). The solutions act to cleanse the colon but patient non-
compliance and other indicators, such as constipation, obesity and cirrhosis of the liver,
can result in unsuccessful viewings of the colon (68). This can result in cancelled
procedures, and added medical cost. In an effort to determine whether the consumption
of Medilac ®, as co-treatment to polyethylene glycol or sodium phosphate solutions,
improved the efficacy of the traditional colon cleansing therapies, Lee et al. conducted a
randomized, single-blinded, active control, clinical trial (n=107).
Patients who presented with constipation as defined by Rome III criteria were
enrolled in the study in parallel to controls that were not constipated but still scheduled
for colonoscopy. For two weeks, the participants who received the probiotic intervention
consumed one capsule orally three times per day. On the night before and on the
morning of the colonoscopy, they consumed the traditional sodium phosphate solution.
Colonoscopy preparation was evaluated immediately after each procedure by
endoscope. The group of constipated patients that received the probiotic had fewer
unsatisfactory scores compared to the constipated patients that received placebo
(45.1% vs 79.2% p<0.0001). There was no difference when comparing normal patients
with constipated ones. Interestingly, the group that received probiotics had lower
incidences of vomiting and bloating related adverse events.
51
The study merits attention as its use of probiotics was paired to enhance a
medical procedure’s effectiveness. The study, however, failed to evaluate symptoms
due to probiotic consumption there may have been a myriad of symptoms that
participants experienced while taking the probiotic that were not recorded. The group
that received the probiotic in the constipated group showed a significantly better total
colon cleansing score when compared to placebo, with the assumption that this is due
to alleviation of the constipation by the probiotic, however no effort was made to record
bowel movement frequency in participants (69). If Lee et al. had used a questionnaire to
evaluate stool frequency, consistency and gastrointestinal symptoms, the impact of B.
subtilis could have been further elucidated. At this point in time, no research evaluating
the effect of Bacillus probiotics in constipated patients has been conducted.
Conclusions
The increasing acceptance and usage of probiotics in different regions of the
world is due to the current state of research which seeks to better understand the
impact of probiotics on human health. Both patients and their physicians are
increasingly finding themselves in situations where they need to decide if a particular
type of probiotic supplement can have a beneficial impact on a disease state. Due to the
increasing costs of health care, and symptoms associated with certain drugs, probiotics
are continuously being turned to as alternative and supplemental health care. It is with
these reasons in mind that the study of probiotics requires a multidisciplinary approach
that evaluates both the microbiological and clinical impacts of probiotic consumption.
This warrants the use of randomized, double-blind, placebo-controlled studies that
utilize validated, health-related quality of life questionnaires, microbial sequencing
techniques and metabolomics to substantiate causal relationships between probiotic
52
usage and health benefits. This type of research will increase the confidence in which
probiotics can be turned to as adjuvant and alternative therapies for disease states.
Table 1-1. Syndrome assignments of the SF-36, PGWB, and GSRS
SF-36 PGWB GSRS
Physical fFunction
Role Limitations-Physical
Bodily Pain
General Health
Mental Health
Role Limitations-Emotional
Vitality
Social Function
Mental Component Summary
Physical Component Summary
Anxiety
Depression
Positive Well-Being
Behavioral Control
General Health
Vitality
Abdominal Pain
Reflux
Indigestion
Constipation
Diarrhea
53
CHAPTER 2 PURPOSE
The purpose of this study is to evaluate the effect of the probiotic B. subtilis
R0179 on gastrointestinal function, general wellness and gastrointestinal viability in
healthy young adults. To date, no studies have been conducted evaluating the dose
response of a probiotic on gastrointestinal function, general wellness and
gastrointestinal viability. The current study aims to fill this gap in the literature by
administering oral doses of the probiotic B. subtilis R0179 to healthy adults for a period
of 4 weeks and to evaluate the development of any general wellness or gastrointestinal
symptoms through the use of a daily questionnaire and the validated gastrointestinal
symptom response scale. It is hypothesized that B. subtilis R0179 will have no effect on
the symptoms reviewed in the questionnaires and that due to its spore forming
capability, B. subtilis R0179 will survive the antimicrobial properties of the human
gastrointestinal tract and pass through to the colon where it can exert probiotic effects.
54
CHAPTER 3 METHODS AND PROCEDURES
Study Design
A six-week, randomized, double-blind, placebo-controlled study was carried out
with 81 healthy adults, 18-50 years of age. Participants were started on the protocol in
five waves consisting of a one week baseline period, a four week intervention period
and a one week washout period (Figure 3-1). Wave 1 was randomized July 30th 2012
(n=21), wave 2 was randomized August 6th (n=17), wave 3 was randomized August 13th
(n=20), wave 4 was randomized August 20th (n=19), and wave 5 was randomized
August 27th (n=4). All participants were randomized in parallel into one of three possible
dosages of the probiotic B. subtilis R0179 (10 billion, 1 billion and 0.1 billion
CFU/capsule) or to the placebo group. Institutional Review Board approval was
obtained from the University of Florida’s IRB-01 on 06/28/2012 for a period of one year
ending on 06/19/2013, this was extended to 6/20/2014 for further data analysis
(Appendix A).
Inclusion and Exclusion Criteria before Attaining Consent
Participants were included in the study if they fell within the age range of 18-50
years. They were required to be willing and able to complete the Informed Consent
(Appendix B) in English as well as completing a Global Physical Activity Questionnaire
(GPAQ) and being determined moderately active by the GPAQ (Appendix C).
Moderately active was defined as having to complete at least sixty minutes and at most
three hundred minutes of moderate to vigorous intensity exercise(s) per week. Any
participants who fell outside of these parameters were excluded from participating in the
study. These parameters were selected as recommended by the American College of
55
Sports Medicine in a position stand published in 1998 (70) Participants were required to
be willing to take height and weight measurements as well as providing demographic
information. They were required to be willing to consume the B. subtilis R0179 (at
approximate doses of 0.1, 1.0, and 10 billion CFU/capsule/day) or a placebo for a 28
day period. They were required to have Internet access for the duration of the study in
order to complete a daily questionnaire electronically (Appendix D). They were also
required to complete the Gastrointestinal Symptom Response Survey (GSRS) three
times throughout the study: once in the baseline week, once in week 4 of the
intervention period and once after the washout period (Appendix E). In addition, they
were required to provide three stool samples (baseline, week 4 of intervention, and
during washout).
Participants were excluded from the study if they did not meet any of the above
criteria and if they were taking any medications for constipation or diarrhea. They were
excluded if they had taken antibiotics within the past four weeks prior to randomization
or if they were already taking a probiotic supplement that they did not want to
discontinue at least two weeks prior to the beginning of the study. In addition, they were
excluded if they had or were currently being treated for any diseases or illnesses such
as the gastrointestinal diseases: gastric ulcers, Crohn’s disease and ulcerative colitis or
other chronic diseases such as diabetes and kidney disease or immune-compromising
diseases/conditions such as HIV, AIDS, hepatitis, and cancer, or if they were a
transplant patient.
Recruitment
Healthy adults were recruited by flyers and word of mouth on the University of
Florida campus. Following an initial screening of participants using the inclusion and
56
exclusion criteria listed above, participants were consented by trained study
coordinators and proceeded to take the GPAQ. After completion of the GPAQ, any
participant that completed less than sixty and more than three hundred minutes of
vigorous to moderate intensity exercise per week were notified of their ineligibility.
Recruitment began on 7/02/2012 and ran intermittently until 08/23/2012.
Baseline
Following informed consent, participants were provided with a customized web
URL to their daily questionnaires which were to be completed in the evening before
11:59pm of that day. Participants were also instructed on the proper technique for the
collection of a whole stool sample by trained research staff. They were provided a
commode specimen collection system (Fisher Scientific) and were instructed to bring
the sample in for processing on ice within four hours of defecation. Following the
completion of the baseline week, participants attended their first clinic visit in which
research coordinators administered the GSRS, took weight measurements on a Health
o meter® Professional scale (model: 349KLX) and height measurements on a Seca
Stadiometer (model 213). Participants also completed a demographic information
questionnaire evaluating race and ethnicity. Participants were then randomized to one
of three B. subtilis probiotic groups (0.1, 1 and 10 billion CFU/capsule/day) or placebo.
Trained research staff monitored the completion of the daily questionnaires for
each participant. If the questionnaire had not been completed by 11:59 p.m., an email
reminder was sent to the participant to ensure full compliance to study protocol.
Participants who failed to comply with study protocol were withdrawn from the study
prior to randomization.
57
Randomization and Intervention
A simple randomization of four intervention groups was completed using
Microsoft Excel 2010 (Version 14.0.6123.5001) by a collaborator who did not have
access to study participants or data. No restrictions were implemented in the
randomization sequence. Each random allocation was formatted to fit on a single sheet
of white paper, indicating the coded intervention group, the date, the number of pill
bottles given to the participants and the participant identification number. These were
placed in envelopes and sealed to ensure the integrity of the blinding process. The
envelopes were separated into five stacks, each stack represented a different
randomization date, on top of each envelope was printed: Stack # and Envelope #.
Participants were enrolled by trained research coordinators during the consenting
process. On randomization day research coordinators unsealed the envelopes and
assigned the participants to their intervention group.
Post randomization, participants were instructed to take one capsule per day at
the end of a meal. They were provided with enough capsules distributed between two
pill bottles for the entire 4-week intervention period. The bottles were clearly marked
with a specialized four digit code (for the purposes of randomization) and the
instructions, “Consume one capsule per day at the end of a meal.” Participants were
then instructed on how to open the bottles and that they should begin consumption of
the capsule that day (Day 8 of the study) and to continue doing so for four weeks. They
were also instructed to retain the bottles and any extra capsules. In the last week of the
intervention phase, participants provided their second fecal sample.
All research staff were blinded. Capsules provided by Lallemand Health
Solutions (Montreal, QC) were identical in size, shape and color. The capsules from
58
each intervention group were inside white, unmarked, and sealed pill bottles. Labels for
each intervention group were applied by a separate research lab in order to ensure the
fidelity of the blinding process.
Washout and Post Intervention
Following the intervention phase, participants attended their second clinical visit
where they returned the pill bottles and any remaining capsules. They were
administered a GPAQ and GSRS by study coordinators. Final weight measurements
were taken. Participants were then instructed to continue completing their daily
questionnaires for the next seven days and that they would be administered a final
GSRS on the last day of the washout period. They were also instructed to bring in a
final stool sample immediately following the washout week.
Compensation
Following completion of the entire study: six weeks of completing the daily
questionnaire, consumption of the capsules, and providing three fecal samples
($15USD/stool sample) participants were provided a maximum of $200USD.
Stool Protocol
Stool samples were collected by a commode specimen collection system (Fisher
Scientific) during baseline, intervention (week 4) and post washout to assess the
viability of B. subtilis R0179. Participants were instructed to place samples upon ice
immediately after defecation and to deliver samples to study personnel within four
hours. Samples were then homogenized by kneading in a strong plastic bag.
Approximately 1 g of homogenized stool was added to pre weighed 15 mL conical
tubes. Exact stool weight was obtained by difference and a 1:10 w/v dilution with
phosphate buffered saline (PBS) was agitated with 4 to 5 glass beads (1mm) to break
59
up particles. Samples were centrifuged (Sorval legend RT+) for 2 minutes at 54 rcf to
pellet large particles. Supernatants were gently mixed and 10-fold serial dilutions in PBS
were placed in a water bath at 80°C for 10 minutes to eliminate non spore-forming
organisms. Positive controls demonstrated no loss of viability due to heating. Dilutions
were plated using in duplicate on Luria Bertoni agar (Fisher Scientific, #BP1425-500)
and incubated overnight at 37°C. Spreaders were sterilized by incineration to prevent
contamination from spores.
Statistical Methods
Equivalence Testing
Equivalence testing was conducted for both the Daily Questionnaires (DQ) and
the GSRS. Syndromes and symptoms of the daily questionnaires were tested for
unequal variances using Levene’s test, upon rejection of the null hypothesis (α =0.05)
data was transformed accordingly. Each syndrome of the daily questionnaire was
compared on a between groups and per week basis, with a predetermined specified
practical difference threshold of .377 (approximating a value of one unit on the
questionnaire) after the data was transformation. When mean symptom and syndrome
scores were significant (P<0.05), the null hypothesis was rejected indicating a practical
equivalence between the intervention groups. Each syndrome of the GSRS was
compared on a between group and per intervention period basis, with a predetermined
specified practical difference threshold of 1. When mean syndrome scores were
significant (P<0.05), the null hypothesis was rejected indicating a practical equivalence
between the intervention groups.
Daily Questionnaires evaluated a variety of general wellness symptoms (Table 3-
1 and Table 3-2) that were rated on a scale of 0 (no symptoms present) to 6 (very
60
severe). Means of subject responses were taken for each symptom by week. These
weekly means were then grouped with other similar symptoms to form a mean
syndrome score. The syndromes analyzed were: GI distress, cephalic, ear-nose-throat,
behavioral, emetic, and epidermal. Symptoms that could not be grouped into a
syndrome were evaluated individually. Due to non-normality, square roots of syndrome
scores were taken.
Individual symptoms of the DQ that did not fit into a syndrome grouping were
also tested for equivalence using transformed data. Mean sleep equivalence testing
was conducted at a predetermined specified difference threshold of 30 mins. Mean
bowel movements was tested for equivalence at a predetermined specified practical
difference threshold of .20 bowel movements, bowel movements were pooled into
baseline, intervention (weeks 1-4) and washout scores. Kruskall-Wallis tests were
conducted to evaluate the change in syndrome and symptom scores within intervention
groups over the 6-week study. When significance was reached the pair-wise
comparisons were made using the Wilcoxon test.
Gastrointestinal Symptom Rating Scales were administered to participants at
baseline, week 4 of intervention and post washout. Individual symptoms assessed by
the GSRS were averaged into syndrome scores (Table 3-2), these syndrome scores
were then compared across the baseline, intervention and washout periods between
groups. Kruskall-Wallis tests were conducted to evaluate the change in syndrome
scores within intervention groups over the baseline, intervention and washout periods.
When significance was reached the pair-wise comparisons were made using the
Wilcoxon test.
61
B. subtilis Viability
Viability of B. subtilis R0179, recovered from stool samples, was assessed using one
way ANOVA followed subsequently by Tukey-Kramer HSD when significance was
reached (p<0.05). Data was normalized when appropriate. Data analyzed was log
(CFU/g).
62
Figure 3-1. Study design
63
Table 3-1. Daily Questionnaire syndrome assignments
Gastrointestinal Distress
Cephalic ENT Behavioral Emetic Epidermal Unclassified symptoms
Bloating Headache Sore throat Anxiety Nausea Itching Fatigue
Flatulence Dizziness Runny eyes Depression Vomiting Skin rash Satiety
Stomach noises Nasal congestion Skin redness and flushing
Hours of sleep
Abdominal cramps Blocked ear canal
Constipation
Diarrhea
Bowel movement frequency
64
Table 3-2. Gastrointestinal Symptom Rating Scale syndrome assignments
Abdominal Pain Indigestion Reflux Constipation Diarrhea
Abdominal pain Stomach rumbling Heart burn Constipation Diarrhea
Hunger pain Bloating Acid regurgitation Hard stools Loose stools
Nausea Burping
Increased Flatus
Feelings of incomplete evacuation Urgent need for defecation
65
CHAPTER 4 RESULTS
Participants
Eighty-one participants completed the six week study (Figure 4-1). One
participant in the 10 billion CFU intervention group voluntary withdrew after experiencing
some itching and redness symptoms and another was withdrawn from the study due to
non-compliance to study protocol. One serious adverse event was reported by a
participant randomized to the 0.1 billion CFU/capsule/day intervention, but this
participant did not cease consumption of the probiotic and completed the study.
Participant ages ranged from 20 to 49 across all intervention groups and did not differ in
gender, age, race/ethnicity, BMI categories, physical activity level, questionnaire
compliance and intervention compliance (% total of capsules consumed during
intervention). The 0.1 billion CFU/group differed from the other intervention groups in
the stool compliance category (85% compliance vs. 100, 97 and 90 in the placebo, 0.1
and 10 billion CFU groups respectively, p<0.01) (Table 4-1).
Daily Questionnaire Analysis
Daily Questionnaires (DQ) were administered to participants each night for the
duration of the study, a total of 3402 DQ were completed and analyzed. Syndrome
testing is presented in Table 4-2. Individual symptom data is presented in Table 4-3.
The scores of the gastrointestinal distress syndrome of the DQ (symptoms: bloating,
flatulence, stomach noises and abdominal cramps) of the placebo, 0.1 billion and 10
billion CFU groups were concluded equivalent. The 0.1 billion CFU (0.3±0.1) group was
concluded not equivalent to the 1 billion (0.6±0.1) and 10 billion 0.7±0.1) CFU groups in
the baseline week. A subsequent Kruskall-Wallis test revealed that intervention groups
66
were no different from their mean baseline syndrome scores as the study progressed.
This indicates that intervention scores did not change over time and because all scores
were below the clinically significant threshold of 1 on the questionnaire, the syndrome
scores do not indicate that study participants were negatively reacting to consumption of
B. subtilis R0179 (Figure 4-2).
Similar results were produced for the cephalic syndrome scores (symptoms:
headache, dizziness) when non-equivalence in weeks 2 and 3 of intervention period
were concluded between the 0.1 billion CFU group (0.2±0.1) and the 10 billion CFU
group (0.3±0.1) and between the 0.1 billion CFU group (0.1±0.03) and the placebo
group (0.4±0.1) respectively (Table 4-2). Once again these scores were well below the
clinically significant threshold of 1 (Figure 4-3). A Kruskall-Wallis test confirmed that
intervention group means did not differ from baseline when compared to the intervention
and washout weeks.
The epidermal syndrome scores (symptoms: itching, skin rash, skin
redness/flushing) were concluded equivalent across all intervention by period
comparisons, means ranged from 0.02±0.02 in the 10 billion CFU group to 0.1±0.1 in
the 0.1 billion CFU group (Table 4-2). All syndrome score means were well below the
clinically significant threshold of 1 (Figure 4-4). A Kruskall-Wallis test confirmed that
group means did not differ from baseline when compared to the intervention and
washout weeks.
The ear-nose-throat syndrome scores (symptoms: sore throat, runny eyes, nasal
congestion, blocked ear canal) were concluded equivalent across all period by
intervention group comparisons except for during week 1 of the intervention period
67
where the 0.1 billion CFU group (0.4±0.1) and the 1 billion CFU group (0.1±0.03) groups
were concluded not equivalent (Table 4-2). All syndrome score means were below the
clinically significant threshold of 1 (Figure 4-5). A Kruskall-Wallis test confirmed that
group means did not differ from baseline when compared to the intervention and
washout weeks.
The behavioral syndrome scores (symptoms: anxiety and depression) were
concluded not equivalent for a variety of intervention and period comparisons (Table 4-
2). Scores ranged from 0.1±0.1 in the 0.1 and 1 billion CFU groups to 0.6±0.2 in the
placebo group. All mean syndrome scores were well below the clinically significant
threshold of 1 (Figure 4-6). A Kruskall-Wallis test confirmed that group means did not
differ from baseline when compared to the intervention and washout weeks.
Emetic syndrome scores (symptoms: nausea and vomiting) were concluded
equivalent across all period by intervention comparisons (Table 4-2). Mean scores
ranged from 0.003±0.003 in the 0.1 billion CFU group to 0.1±0.1 in the placebo, 1 billion
and 10 billion CFU groups. These values were well below the clinically significant
threshold of 1 (Figure 4-7). A Kruskall-Wallis test revealed a difference in the 0.1 billion
CFU group, Χ2 (5, N = 21) = 13.3, p = 0.02. Subsequent analysis using the Wilcoxon
method revealed that weeks 2 (p=0.008, 0.1±0.04) and 3 (p=0.02, 0.02±0.02) of the
intervention period and the washout week (p=0.04, 0.1±0.1) were significantly different
from week 1 (0.003±0.003) and that week 4 (0.1±0.03) was significantly different from
weeks 3 (p=0.03) and 2 (p=0.01) (Table 4-2).
Individual symptoms that could not incorporated into a syndrome score were also
tested using the equivalence testing method. The constipation symptom scores were
68
concluded non-equivalent for a variety of period and intervention comparisons (Table 4-
3) Scores ranged from 0.1±0.04 in the placebo group to 0.7±0.3 in the 10 billion CFU
group. All mean constipation scores were below the clinically significant threshold of 1
(Figure 4-8). A Kruskall-Wallis test confirmed that group means did not differ from
baseline when compared to the intervention and washout weeks.
The fatigue symptom also had a variety of non-equivalent period by intervention
group comparisons (Table 4-3). Mean scores ranged from 0.4±0.1 in the 0.1 billion
group to 1.1±0.3 in the placebo group. The placebo group also had a score of 1.0±0.3
during week 3 of the intervention period (Figure 4-9). A Kruskall-Wallis test confirmed
that group means did not differ from baseline values when compared to the intervention
and washout weeks.
The diarrhea symptom scores were concluded equivalent for the baseline week,
and weeks 1 and 2 of the intervention period. Means were concluded not equivalent
between the 1 billion CFU group (0.4±0.1) and the other intervention groups during
week 3 of intervention. During week 4 the 0.1 billion CFU group (0.2±0.1) and the 10
billion CFU group (0.1±0.1) were concluded equivalent; they were concluded not
equivalent when compared to the placebo (0.4±0.1) and 1 billion CFU (0.3±0.1) groups.
During the washout week, the 0.1 billion CFU group (0.1±0.04) was not equivalent to
the other intervention groups mainly because its mean was lower than the others (Table
4-3). All diarrhea symptom scores were below the clinically significant score of 1 (Figure
4-10). A Kruskall-Wallis test confirmed that group means did not differ from baseline
when compared to the intervention and washout weeks.
69
Bowel movement frequency was recorded daily using the daily questionnaire.
Mean bowel movement frequency ranged from 1.4±0.1 in the placebo group to 1.6 in
the 10 billion CFU group (Figure 4-11). A variety of period and intervention group
comparisons were concluded not equivalent (Table 4-3). A Kruskall-Wallis test
confirmed that bowel movement frequency did not significantly change when comparing
baseline means to intervention and washout values.
Hours of sleep were compared using the equivalence testing method. Means
ranged from 6.5±0.2 in the placebo group to 7.3 in the 1 billion CFU group (Figure 4-
12). A variety of period and intervention group comparisons were concluded not
equivalent (Table 4-3). A Kruskall-Wallis test confirmed that mean hours of sleep did not
significantly change when comparing baseline means to intervention and washout
values.
Gastrointestinal Symptom Rating Scale Analysis
A total of 243 GSRS questionnaires were completed by participants. The
abdominal pain syndrome (symptoms: abdominal pain, hunger pains and nausea)
(Figure 4-13), reflux syndrome (symptoms: heart burn and acid regurgitation) (Figure 4-
14), diarrhea syndrome (diarrhea, loose stools and urgent need for defecation) (Figure
4-15), indigestion syndrome (stomach rumbling, abdominal distension, eructation and
increased flatus) (Figure 4-16), constipation syndrome (constipation, hard stools and
feeling of incomplete evacuation) (Figure 4-17) were significant across all intervention
groups and periods indicating equivalence. Equivalence testing for the GSRS
questionnaire is presented in Table 4-4.
70
Viability of B. subtilis R0179 in Humans
Least square means estimates of enumerate plate counts of B. subtilis showed a
dramatic increase in the amount of viable bacteria between weeks 1, (baseline), 5
(intervention) and 6 (washout) (Table 4-5). All intervention groups showed significant
increases in bacteria counts presented as log CFU/g +1 and the placebo group was not
significant (Figure 4-18). Increases occurred in week 5, the end of the intervention
period, and diminished by the final fecal collection when probiotic consumption ceased.
71
Figure 4-1. Participant flow diagram
72
Table 4-1. Participant demographics
Placebo (n=20)
0.1 billion CFU (n=21)
1 billion CFU (n=20)
10 billion CFU (n=20)
P Value1
Gender (M/F), n 9/11 8/13 12/8 10/10 NS
Age, years Median (range) 23 (20-46) 23 (20-49) 22 (20-31) 23 (19-46) NS
Race/ethnicity2,n (%) NS
Asian 2 (10) 7 (33) 2 (10) 2 (10)
Black 4 (20) 2 (10) 2 (10) 2 (10)
Hispanic 3 (15) 2 (10) 3 (15) 2 (10)
White 9 (45) 9 (43) 13 (65) 14 (70)
Other 2 (10) 1 (4) 1 (5) -
BMI percentiles, n (%) NS
<85th (healthy weight) 10 (50) 12 (60)3 13 (65) 9 (45)
85th-94th (overweight) 6 (30) 7 (35) 4 (20) 9 (45)
≥95th (obese) 4 (20) 1 (5) 3 (15) 2 (10)
Activity Level ± SEM NS
Baseline 185.5 ± 15.9 197.3 ± 18.1 168.3 ± 16.7 199.0 ± 13.0
Washout 161.8 ± 19.9 206.3 ± 36.1 178.5 ± 28.8 234.0 ± 29.8
Compliance (%)
Questionnaire Protocol 99 98 99 97 NS
Intervention Protocol 95 89 94 93 NS
Stool Protocol 100 97 85* 90 0.01
73
Table 4-2. Daily Questionnaire syndrome equivalence testing
Syndrome Period 0.1 billion CFU 1 billion CFU 10 billion CFU Placebo
Gastrointestinal Distress
Baseline 0.3 (± 0.1) a 0.6 (± 0.1)
b 0.7 (± 0.1)
bc 0.5 (± 0.8)
abc
Week 1 0.4 (± 0.1) a 0.6 (± 0.1)
b 0.5 (± 0.1)
abc 0.5 (± 0.1)
abc
Week 2 0.3 (± 0.1) a 0.6 (± 0.1)
b 0.6(± 0.1)
b 0.6 (± 0.1)
b
Week 3 0.3 (± 0.01) a 0.7 (± 0.1)
b 0.7 (± 0.1)
bc 0.6 (± 0.1)
bc
Week 4 0.3 (± 0.1) a 0.7 (± 0.1)
b 0.5 (± 0.1)
bc 0.6 (± 0.1)
bc
Washout 0.3 (± 0.1) a 0.6 (± 0.1)
b 0.4 (± 0.1)
abc 0.5 (± 0.1)
bc
Cephalic Baseline 0.2 (± 0.1) 0.2 (± 0.04) 0.2 (± 0.1) 0.2 (± 0.1)
Week 1 0.2 (± 0.1) 0.1 (± 0.04) 0.2 (±0.1) 0.2 (± 0.1)
Week 2 0.2 (± 0.1) a 0.1 (± 0.04)
ab 0.3 (±0.1)
c 0.2 (± 0.1)
abc
Week 3 0.1 (± 0.03) a 0.2 (± 0.1)
ab 0.3 (±0.1)
abc 0.4 (± 0.1)
bc
Week 4 0.2 (± 0.1) 0.2 (± 0.1) 0.2 (±0.1) 0.2 (± 0.1)
Washout 0.3 (± 0.1) 0.2 (± 0.1) 0.2 (±0.1) 0.2 (± 0.1)
Data presented as mean (± std. error). Intervention by week combinations not connected by the same letter were concluded as not equivalent (p>0.05). Rows with no superscripts were concluded equivalent (p<0.05)
74
Table 4-2. Continued
Syndrome Period 0.1 billion CFU 1 billion CFU 10 billion CFU Placebo
Epidermal Baseline 0.1 (± 0.1) 0.01 (± 0.01) 0.03 (± 0.01) 0.1 (± 0.02)
Week 1 0.02 (± 0.01) 0.04 (± 0.02) 0.04 (± 0.02) 0.1 (± 0.04)
Week 2 0.1 (± 0.1) 0.1 (± 0.04) 0.05 (± 0.03) 0.04 (± 0.03)
Week 3 0.02 (± 0.01) 0.1 (± 0.1) 0.04 (± 0.02) 0.1 (± 0.04)
Week 4 0.02 (± 0.01) 0.02 (± 0.02) 0.02 (± 0.02) 0.1 (± 0.04)
Washout 0.02 (± 0.02) 0.01 (± 0.01) 0.02 (± 0.02) 0.1 (± 0.04)
Ear-Nose-Throat Baseline 0.1 (± 0.4) 0.1 (± 0.04) 0.1 (± 0.04) 0.2 (± 0.1)
Week 1 0.4 (± 0.1) a 0.1 (± 0.03)
b 0.1 (± 0.04) 0.3 (± 0.2)
Week 2 0.2 (± 0.1) 0.2 (± 0.1) 0.2 (± 0.1) 0.3 (± 0.1)
Week 3 0.2 (± 0.1) 0.2 (± 0.1) 0.2 (± 0.1) 0.4 (± 0.2)
Week 4 0.2 (± 0.1) 0.2 (± 0.1) 0.2 (± 0.1) 0.3 (± 0.1)
Washout 0.2 (± 0.1) 0.2 (± 0.1) 0.1 (± 0.1) 0.2 (± 0.1)
Data presented as mean (± std. error). Intervention by week combinations not connected by the same letter were concluded as not equivalent (p>0.05). Rows with no superscripts were concluded equivalent (p<0.05).
75
Table 4-2. Continued
Syndrome Period 0.1 billion CFU 1 billion CFU 10 billion CFU Placebo
Behavioral Baseline 0.2 (± 0.1) a 0.2 (± 0.1)
b 0.5 (± 0.1)
c 0.5 (± 0.1)
c
Week 1 0.2 (± 0.1) a 0.1 (± 0.1)
a 0.6 (± 0.2)
b 0.4 (± 0.1)
b
Week 2 0.1 (± 0.1) a 0.1 (± 0.1)
a 0.5 (± 0.1)
b 0.4 (± 0.2)
b
Week 3 0.1 (± 0.1) a 0.2 (± 0.01)
ab 0.5 (± 0.1)
c 0.4 (± 0.2)
bc
Week 4 0.2 (± 0.1) a 0.1 (± 0.1)
a 0.5 (± 0.2)
b 0.6 (± 0.2)
b
Washout 0.3 (± 0.2) a 0.2 (± 0.1)
a 0.5 (± 0.2)
b 0.4 (± 0.1)
b
Emetic Baseline 0.003 (± 0.003) 0.1 (± 0.1) 0.1 (± 0.2) 0.05 (± 0.04)
Week 1 0.1 (± 0.04) 0.1 (± 0.04) 0.03 (± 0.01) 0.1 (± 0.1)
Week 2 0.1 (± 0.1) 0.1 (± 0.1) 0.1 (± 0.1) 0.1 (± 0.04)
Week 3 0.02 (± 0.02) 0.1 (± 0.1) 0.1 (± 0.04) 0.1 (± 0.1)
Week 4 0.1 (± 0.03) 0.1 (± 0.1) 0.03 (± 0.02) 0.1 (± 0.1)
Washout 0.1 (± 0.1) 0.1 (± 0.1) 0.08 (± 0.04) 0.1 (± 0.1)
Data presented as mean (± std. error). Intervention by week combinations not connected by the same letter were concluded as not equivalent (p>0.05). Rows with no superscripts were concluded equivalent (p<0.05).
76
Figure 4-2. Gastrointestinal distress syndrome
0
1
2
3
4
5
6
Baseline Week 1 Week 2 Week 3 Week 4 Washout
Mean S
yndro
me S
core
Gastrointestinal Distress
Placebo 0.1 billion 1 billion 10 billion
77
Figure 4-3. Cephalic syndrome
0
1
2
3
4
5
6
Baseline Week 1 Week 2 Week 3 Week 4 Washout
Mean S
yndro
me S
core
Cephalic
Placebo 0.1 billion 1 billion 10 billion
78
Figure 4-4. Epidermal syndrome
0
1
2
3
4
5
6
Baseline Week 1 Week 2 Week 3 Week 4 Washout
Mean S
yndro
me S
core
Epidermal
Placebo 0.1 billion 1 billion 10 billion
79
Figure 4-5. Ear nose and throat syndrome
0
1
2
3
4
5
6
Baseline Week 1 Week 2 Week 3 Week 4 Washout
Mean S
yndro
me S
core
Ear Nose and Throat
Placebo 0.1 billion 1 billion 10 billion
80
Figure 4-6. Behavioral syndrome
0
1
2
3
4
5
6
Baseline Week 1 Week 2 Week 3 Week 4 Washout
Mean S
yndro
me S
core
Behavrioal
Placebo 0.1 billion 1 billion 10 billion
81
Figure 4-7. Emetic syndrome
0
1
2
3
4
5
6
Baseline Week 1 Week 2 Week 3 Week 4 Washout
Mean S
yndro
me S
core
Emetic
Placebo 0.1 billion 1 billion 10 billion
82
Table 4-3. Daily Questionnaire symptom equivalence testing
Syndrome Period 0.1 billion CFU 1 billion CFU 10 billion CFU Placebo
Constipation Baseline 0.1 (± 0.1) a 0.4 (± 0.2) b 0.4 (± 0.2) c 0.2 (± 0.1) a
Week 1 0.2 (± 0.1) a 0.3 (± 0.1) b 0.7 (± 0.3) c 0.1 (± 0.04) a
Week 2 0.2 (± 0.01) a 0.3 (± 0.1) b 0.6 (± 0.2) c 0.3 (± 0.1) b
Week 3 0.1 (± 0.1) a 0.6 (± 0.2) ab 0.7 (± 0.3) b 0.3 (± 0.1) c
Week 4 0.1 (± 0.1) a 0.5 (± 0.1) b 0.7 (± 0.2) c 0.2 (± 0.1) a
Washout 0.1 (± 0.03) a 0.5 (± 0.2) b 0.5 (± 0.2) c 0.3 (± 0.1) bc
Diarrhea Baseline 0.1 (± 0.04) 0.2 (± 0.1) 0.2 (± 0.1) 0.2 (± 0.1)
Week 1 0.2 (± 0.03) 0.2 (± 0.1) 0.1 (± 0.03) 0.2 (± 0.1)
Week 2 0.1 (± 0.1) 0.3 (± 0.1) 0.3 (± 0.1) 0.2 (± 0.1)
Week 3 0.1 (± 0.1) a 0.4 (± 0.1)b 0.3 (± 0.1) a 0.2 (± 0.1) a
Week 4 0.2 (± 0.1) a 0.3 (± 0.1) b 0.1 (± 0.1) a 0.4 (± 0.1) b
Washout 0.1 (± 0.04) a 0.2 (± 0.1) b 0.2 (± 0.1) b 0.3 (± 0.1) b
Fatigue Baseline 0.7 (± 0.2) a 0.7 (± 0.2) a 0.8 (± 0.2) b 1.1 (± 0.3) b
Week 1 0.7 (± 0.2) a 0.5 (± 0.2) a 0.8 (± 0.2) b 0.9 (± 0.3) b
Week 2 0.6 (± 0.2) a 0.7 (± 0.2) b 0.9 (± 0.2) c 0.9 (± 0.3) d
Week 3 0.4 (± 0.1) a 0.6 (± 0.2) b 0.9 (± 0.3) c 1.0 (± 0.3) d
Week 4 0.5 (± 0.1) a 0.6 (± 0.2) ab 0.8 (± 0.2) c 0.8 (± 0.3) b
Washout 0.5 (± 0.2) a 0.5 (± 0.2) a 0.7 (± 0.2) c 0.6 (± 0.3) ac
Data presented as mean (± std. error). Intervention by week combinations not connected by the same letter were concluded as not equivalent (p>0.05). Rows with no superscripts were concluded equivalent (p<0.05).
83
Table 4-3. Continued
Syndrome Period 0.1 billion CFU 1 billion CFU 10 billion CFU Placebo
Satiety Baseline 0.7 (± 0.2) a 0.9 (± 0.2) a 0.9 (± 0.2) b 0.8 (± 0.2) b
Week 1 0.3 (± 0.1) a 0.8 (± 0.2) b 1.1 (± 0.3) c 0.7 (± 0.2) d
Week 2 0.3 (± 0.1) a 0.8 (± 0.2) b 0.9 (± 0.2) c 0.9 (± 0.3) c
Week 3 0.3 (± 0.1) a 0.8 (± 0.2) b 1.0 (± 0.3) b 0.9 (± 0.3) c
Week 4 0.4 (± 0.1) a 0.9 (± 0.3) b 0.9 (± 0.3) b 0.9 (± 0.3) c
Washout 0.3 (± 0.1) a 0.9 (± 0.3) b 0.8 (± 0.3) c 0.8 (± 0.3) c
Bowel Movements
Baseline 1.5 (± 0.2) ab 1.3 (± 0.1) a 1.5 (± 0.1) ab 1.55 (± 0.2) ab
Intervention (Weeks 1-4)
1.5 (± 0.1) ab 1.5 (± 0.1) ab 1.5 (± 0.1) ab 1.4 (± 0.1) a
Washout 1.4 (± 0.1) a 1.4 (± 0.1) b 1.6 (± 0.2) a 1.5 (± 0.2) c
Hours of Sleep
Baseline 6.6 (± 0.2) a 7.1 (± 0.1) b 6.6 (± 0.2) a 6.8 (± 0.2) c
Week 1 6.8 (± 0.2) a 7.1 (± 0.2) b 6.9 (± 0.2) c 6.7 (± 0.1) d
Week 2 6.8 (± 0.2) a 7.1 (± 0.2) b 6.7 (± 0.2) c 6.5 (± 0.2) d
Week 3 7.0 (± 0.2) a 7.3 (± 0.2) a 6.8 (± 0.2) b 6.6 (± 0.2) c
Week 4 7.0 (± 0.2) a 6.9 (± 0.2) a 6.9 (± 0.2) b 6.8 (± 0.2) c
Washout 7.0 (± 0.2) a 7.1 (± 0.1) b 6.7 (± 0.2) a 6.8 (± 0.2) c
Data presented as mean (± std. error). Intervention by week combinations not connected by the same letter were concluded as not equivalent (p>0.05).
84
Figure 4-8. Constipation symptom
0
1
2
3
4
5
6
Baseline Week 1 Week 2 Week 3 Week 4 Washout
Mean S
ym
pto
m S
core
Constipation
Placebo 0.1 billion 1 billion 10 billion
85
Figure 4-9. Diarrhea symptom
0
1
2
3
4
5
6
Baseline Week 1 Week 2 Week 3 Week 4 Washout
Mean S
ym
pto
m S
core
Diarrhea
Placebo 0.1 billion 1 billion 10 billion
86
Figure 4-10. Fatigue symptom
0
1
2
3
4
5
6
Baseline Week 1 Week 2 Week 3 Week 4 Washout
Mean S
ym
pto
m S
core
Fatigue
Placebo 0.1 billion 1 billion 10 billion
87
Figure 4-11. Bowel movement frequency
0
1
2
3
4
5
6
7
8
9
10
Baseline Intervention Washout
Mean B
ow
el M
ovem
ents
Bowel Movement Frequency
Placebo 0.1 billion 1 billion 10 billion
88
Figure 4-12. Hours of sleep
0
2
4
6
8
10
12
Baseline Week 1 Week 2 Week 3 Week 4 Washout
Mean H
ours
of S
leep
Hours of Sleep
Placebo 0.1 billion 1 billion 10 billion
89
Table 4-4. Gastrointestinal Symptom Rating Scale equivalence testing
Syndrome Period 0.1 billion CFU 1 billion CFU 10 billion CFU Placebo
Abdominal Pain Baseline 1.3 (± 0.1) 1.4 (± 0.1) 1.5 (± 0.2) 1.6 (± 0.1)
Intervention 1.5 (± 0.1) 1.6 (± 0.1) 1.6 (± 0.1) 1.8 (± 0.1)
Washout 1.6 (± 0.2) 1.6 (± 0.1) 1.3 (± 0.1) 1.7 (± 0.1)
Reflux Baseline 1.3 (± 0.2) 1.1 (± 0.1) 1.1 (± 0.1) 1.3 (± 0.1)
Intervention 1.3 (± 0.2) 1.1 (± 0.1) 1.2 (± 0.1) 1.3 (± 0.1)
Washout 1.3 (± 0.2) 1.1 (± 0.1) 1.2 (± 0.1) 1.3 (± 0.1)
Indigestion Baseline 1.4 (± 0.1) 1.5 (± 0.01) 1.7 (± 0.1) 1.8 (± 0.1)
Intervention 1.6 (± 0.1) 1.9 (± 0.1) 1.6 (± 0.1) 1.9 (± 0.2)
Washout 1.3 (± 0.1) 1.6 (± 0.1) 1.7 (± 0.1) 1.9 (± 0.1)
Constipation Baseline 1.1 (± 0.1) 1.4 (± 0.1) 1.4 (± 0.1) 1.5 (± 0.1)
Intervention 1.2 (± 0.1) 1.6 (± 0.2) 1.8 (± 0.2) 1.6 (± 0.1)
Washout 1.2 (± 0.1) 1.6 (± 0.2) 1.7 (± 0.2) 1.5 (± 0.1)
Diarrhea Baseline 1.2 (± 0.1) 1.3 (± 0.2) 1.4 (± 0.1) 1.3 (± 0.1)
Intervention 1.2 (± 0.1) 1.6 (± 0.2) 1.5 (± 0.1) 1.7 (± 0.2)
Washout 1.3 (± 0.1) 1.4 (± 0.2) 1.3 (± 0.1) 1.8 (± 0.2)
Data presented as mean (± std. error). Intervention by week combinations were all concluded equivalent (p<0.05).
90
Figure 4-13. GSRS abdominal pain syndrome
1
2
3
4
5
6
7
Baseline Intervention Washout
Mean S
yndro
me S
core
Abdominal Pain
Placebo 0.1 billion 1 billion 10 billion
91
Figure 4-14. GSRS reflux syndrome
1
2
3
4
5
6
7
Baseline Intervention Washout
Mean S
yndro
me S
core
Reflux
Placebo 0.1 billion 1 billion 10 billion
92
Figure 4-15. GSRS indigestion syndrome
1
2
3
4
5
6
7
Baseline Intervention Washout
Mean S
yndro
me S
core
Indigestion
Placebo 0.1 billion 1 billion 10 billion
93
Figure 4-16. GSRS constipation syndrome
1
2
3
4
5
6
7
Baseline Intervention Washout
Mean C
onstipation S
core
Constipation
Placebo 0.1 billion 1 billion 10 billion
94
Figure 4-17. GSRS diarrhea syndrome
1
2
3
4
5
6
7
Baseline Intervention Washout
Mean S
yndro
me S
core
Diarrhea
Placebo 0.1 billion 1 billion 10 billion
95
Table 4-5. Gastrointestinal viability of Bacillus spores
Period Placebo 0.1 billion CFU 1 billion CFU 10 billion CFU
log CFU/g Baseline 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 1.2 ± 0.2
Intervention 1.1 ± 0.1b 4.6 ± 0.1a 5.6 ± 0.1a 6.4 ± 0.1a
Washout 1.6 ± 0.1ab 1.3 ± 0.2ab 0.8 ± 0.1a 2.1 ± 0.1b
Data presented as mean (± std. error). Intervention by week combinations were all concluded equivalent (p<0.05).
Figure 4-18. Gastrointestinal viability presented as means ± SEM
0
1
2
3
4
5
6
7
Baseline Intervention Washout
Log10 C
FU
/G
Gastrointestinal Viability
Placebo 0.1 billion 1 billion 10 billion
96
CHAPTER 5 DISCUSSION AND CONCLUSIONS
Discussion
The spore forming bacteria Bacillus has long been thought to live its life cycle in
the soil, however recovery of vegetative cells from soil samples has been challenging
(71). Fecal samples collected from healthy adults consuming a western diet typically
contain about 104 spores of Bacillus / g of feces. This data suggests that Bacillus
species are natural residents of the human GIT and their residence in soil only serves
as a reservoir for the spore form (62). The use of Bacillus species as probiotics has also
been demonstrated in several studies demonstrating their ability to act as competitive
exclusion agents (48, 72), production of antimicrobials (72), and immunostimulatory
properties (73). All of these properties have been demonstrated in B. subtilis though to
date a clinical evaluation of B. subtilis on general wellness and gastrointestinal health as
a single probiotic agent has not been conducted. The continued use of Bacillus species
in foods and as supplements requires its evaluation at typical therapeutic dosages (i.e.
greater than 0.1 billion CFU/capsule/day). It is with this in mind that a double-blind,
placebo-controlled, randomized, clinical trial (n=81) was conducted to evaluate the
effects of Bacillus subtilis R0179 consumption on general wellness, gastrointestinal
function and gastrointestinal viability in healthy adults. Participants were randomized to
one of four treatment groups (0.1, 1, 10 billion CFU/capsule/day or placebo) for a period
of four weeks and general wellness as well as gastrointestinal symptoms were
evaluated using questionnaires. Stool samples were collected to assess the
gastrointestinal transit viability and impact on the intestinal microbiome of the B. subtilis
R0179.
97
Throughout this six week clinical trial, two participants reported adverse effects
while consuming the probiotic. One participant in the 0.1 billion CFU treatment group
experienced diarrheal symptoms and completed the study, this event did not occur in
any other participants and did not persist. A participant in the 10 billion CFU treatment
group experienced itchiness and redness symptoms that were sporadic and persisted
for several days. The participant voluntarily withdrew from the study, these symptoms
did not occur in any other participants. A third participant reported a serious adverse
event of hospitalization with an admitting diagnosis of hypotension, but did not
discontinue probiotic consumption and completed the study. Despite these adverse
effects, the probiotics were well tolerated by a majority of the study participants.
Questionnaire data on general wellness and gastrointestinal symptom data was
evaluated using equivalence testing. The validated GSRS questionnaire was used to
evaluate gastrointestinal symptoms (42). Mean scores for the abdominal pain, reflux,
indigestion, constipation and diarrhea syndromes for each treatment group (0.1, 1, 10
billion CFU/capsule/day and placebo) across each treatment period (baseline,
treatment, washout) were all below the clinically significant score of 2 (slight discomfort)
on the GSRS. Equivalence testing revealed that all treatment group means across each
treatment period were concluded equivalent.
Daily questionnaires were used to evaluate general wellness in study
participants. Mean scores for the GI distress, cephalic, ENT, behavioral, emetic, and
epidermal syndromes for each treatment group across each week of the study were all
below the clinically significant score of on the DQ. The ungrouped symptoms,
constipation and diarrhea were also below the clinically significant score of 1 on the DQ.
98
The only symptom that exceeded the clinical threshold was the fatigue symptom by the
placebo group during the baseline week and week 3 of the intervention period.
Evaluations of mean hours of sleep and mean bowel movement frequency revealed that
participants were only getting between 6.5 and 7.3 hours of sleep per night and were
averaging 1.4 to 1.6 bowel movements per day. Participants in the placebo group who
were getting the least amount of sleep also reported higher fatigue scores. Conversely,
all treatment groups averaged more than one bowel movement per day which is further
confirmed by their low constipation and diarrhea symptom scores.
Equivalence testing of syndrome and symptom scores revealed a number of
treatment group and period combinations that were concluded were not equivalent,
however no clear trends could be identified. This is particularly clear when intervention
groups were concluded not equivalent in the baseline week and this non-equivalence
continued well into the intervention and washout periods. Kruskall-Wallis tests
confirmed that intervention groups did not change from their baseline values.
Furthermore, when conducting these statistical analyses a very conservative
predetermined practical difference threshold of one unit on the questionnaire was used
in order to ensure non-equivalence could be detected. If that pre-determined practical
difference threshold is tested again at two units on the DQ, then all previously non-
equivalent results are concluded as equivalent.
This use of equivalence testing on symptom data is the first of its kind to be used
in a clinical trial. Standards of care are continually being redefined due to advances in
medicine. Measuring the efficacy of novel therapies against traditional therapies is one
method used to encourage new standards of care. These comparative studies evaluate
99
the superiority of one therapy over another. The opposite is true of equivalence tests,
also referred to as two-one sided tests (TOST). When testing for equivalence, such as
when comparing multiple dosages, traditional hypotheses testing methods are reversed.
A non-significant value, of a traditional comparative test, does not prove that two
treatments are equivalent, it only proves that the treatments may be equivalent although
equivalence is usually not well-defined in superiority testing. For example, a -3%
difference in the eradication rate of H. pylori infection, when comparing a new therapy to
a traditional therapy, may not be significantly different but this does not mean that they
are equivalent, the new therapy is less effective and thus should not be recommended
to patients (74).
A study conducted by Barker et al.2002 evaluated the results of traditional two-
sided tests to that of the TOST using National Immunization Survey data of the number
of vaccinations that while children used when compared to different ethnicities. Nine out
of twenty-one comparisons were contradictory when using the different comparison
methods (75). This disparity is further exacerbated by the fact that superiority testing
gives no indication of how similar differing treatments are only that one is superior or
that it cannot be proven to be superior with the current set of data. The TOST provides
a method by which the similarity of treatment groups can be compared according to a
pre-specified clinically relevant difference threshold. It is for this reason that we set our
practical difference threshold to one-unit when conducting equivalence tests.
This method has demonstrated that there were some non-equivalent values
when comparing mean syndrome and symptom scores, but the scores were no different
from their base line values. Additionally, syndrome and symptom means were below
100
clinically-different threshold values and so, consumption of B. subtilis R0179 does not
have a negative impact on gastrointestinal health and general wellness symptoms at
dosages up to 10 billion CFU/capsule/day. While the safety of B. subtilis R0179 has
already been demonstrated by over 100 clinical trials as reviewed in Tompkins et al.
2010, Chen et al. 2012, Liu et al. 2013, and Hu et al. 2013 until now the effect of R0179
consumption on gastrointestinal and general wellness has never been fully evaluated
especially at dosages higher than 0.1 billion CFU/day.
Analysis of the viability and recovery of the ingested spores from fecal samples
indicated a clear dose response among the treatment groups. Each group was
significantly different from one another with the most spores being recovered from the
10 billion CFU/capsule/day treatment group and the least being recovered from the
placebo group. Furthermore, our results complement data reported by Hong et al. 2009
and Tompkins et al. 2010 which indicate that Bacillus spores transiently colonize the
human gastrointestinal tract and have low adhesive potential
Conclusions
This is the first assessment of Bacillus subtilis R0179 as a single probiotic agent
in a double-blind, placebo-controlled, randomized, clinical trial. Consumption of the
bacterial strain for a period of six weeks did not produce any persisting adverse effects.
Recovery of the probiotic in fecal samples demonstrated a dose response relationship,
and leads further credence to the transitory nature of the bacteria in the human
gastrointestinal tract. The results of this study indicate that the regular consumption of
the B. subtilis R0179 is safe and well tolerated in healthy adults even at high dosages
not typically found in probiotic supplements.
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APPENDIX A IRB APPROVAL
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APPENDIX B INFORMED CONSENT
INFORMED CONSENT FORM to Participate in Research, and
AUTHORIZATION to Collect, Use, and Disclose Protected
Health Information (PHI)
INTRODUCTION
Name of person seeking your consent: Place of employment & position: Please read this form which describes the study in some detail. A member of the research team will describe this study to you and answer all of your questions. Your participation is entirely voluntary. If you choose to participate you can change your mind at any time and withdraw from the study. You will not be penalized in any way or lose any benefits to which you would otherwise be entitled if you choose not to participate in this study or to withdraw. If you have questions about your rights as a research subject, please call the University of Florida Institutional Review Board (IRB) office at (352) 273-9600.
GENERAL INFORMATION ABOUT THIS STUDY
1. Name of Participant ("Study Subject") ___________________________________________________________________
2. What is the Title of this research study?
Evaluation of Bacillus subtilis R0179 in capsules on gastrointestinal survival, gastrointestinal symptoms and general wellness in healthy young adults.
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3. Who do you call if you have questions about this research study?
Principal Investigator: Dr. Wendy Dahl Work: 352-392-1991 ext. 224 Cell: 352-226-1773 Home: 352-374-7798 email: [email protected]
4. Who is paying for this research study?
The sponsor of this study is Institut Rosell Inc.
5. Why is this research study being done?
The purpose of this research study is to determine the survival of three doses of the probiotic B. subtilis (Bacillus subtilis R0179) delivered in capsules on survival through the gastrointestinal tract, impact on intestinal microbes, gastrointestinal symptoms and general well-being in healthy adults age 18 to 50.
You are being asked to be in this research study because you are a healthy adult between the age of 18 and 50 and meet the inclusion and exclusion criteria.
WHAT CAN YOU EXPECT IF YOU PARTICIPATE IN THIS STUDY?
6. What will be done as part of your normal clinical care (even if you did not participate in this research study)?
This study is not related to your normal clinical care.
7. What will be done only because you are in this research study? If you decide to take part in this study, following informed consent, you will begin a 7 day baseline period completing a daily questionnaire (paper or electronic) about bowel movement frequency and gastrointestinal symptoms. The questions will ask you about your bowel movements and gastrointestinal discomfort. Next, you will be randomized to receive the probiotic B. subtilis or placebo and begin taking one capsule per day for 28 days. At the randomization appointment you will be asked to complete the daily questionnaire, global physical activity questionnaire and the gastrointestinal symptom response scale, and height and weight will be determined. You will also continue the daily questionnaire for the 28 days of treatment. If you decide to take part in this study, you will be randomly assigned (much like the flip of a coin) to receive either [study substance] or placebo. A placebo is a substance that looks like and is given in the same way as an experimental treatment but contains no medicine, for example [a sugar pill, an injection of saline (salt water)]. A placebo is used in research studies to show what effect a treatment has compared with taking nothing at
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all. If you are assigned to receive placebo, you will not receive the benefits of the [study substance], if there are any, nor will you be exposed to its risks, which are described below under "What are the possible discomforts and risks?" Studies have shown, however, that about 1 in 3 persons who take a placebo do improve, if only for a short time. You and the physician and other persons doing the study will not know whether you are receiving placebo or [study substance], but that information is available if it is needed. Also, you will have a 50% chance of receiving [study substance] and a 50% chance of receiving placebo. In the remainder of the description of what will be done, both the [study substance] and the placebo will be called "study treatment."
You will be randomized to one of the following groups:
Group 1: B. subtilis R0179 in capsules (approximately 10 billion colony forming units/serving) for period of 28 days.
Group 2: B. subtilis R0179 in capsules (approximately 1 billion colony forming units/serving), for a period of 28 days.
Group 3: B. subtilis R0179 in capsules (approximately 0.1 billion colony forming units/serving), for a period of 28 days.
Group 4: Placebo capsules for a period of 28 days.
In addition, consented participants in each group will be asked to collect one fecal sample at baseline, one fecal sample in week 4 and an additional sample after 7 days following the treatment and drop off to the Food Science and Human Nutrition building within 4 hours of collection. Participants will be provided with specialized plastic containers that can be placed over the toilet seat for fecal collection at home or in the clinical lab restroom facility.
In addition to fecal sample drop off dates, you will be expected to come to the clinical lab in the Food Science and Human Nutrition building for a final visit. At the final appointment you will have your weight measured and you will complete the general wellness questionnaire, GPAQ and GSRS again. Once the intervention period is over, you will be asked to complete a final GSRS on or about 7 days post treatment and continue with the daily questionnaire for 7 days post treatment.
If you have any questions now or at any time during the study, please contact one of the research team members listed in question 3 of this form.
8. How long will you be in this research study?
The total amount of time you will spend participating in the study is 42 days.
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9. How many people are expected to take part in this research study?
Eighty people will be participating in this research study.
WHAT ARE THE RISKS AND BENEFITS OF THIS STUDY AND WHAT ARE YOUR OPTIONS?
10. What are the possible discomforts and risks from taking part in this research study?
Some people may feel uncomfortable when body weight is measured.
Some study participants may feel uncomfortable answering questions about their stool habits.
Some study participants may feel uncomfortable collecting stool samples.
Participation in more than one research study or project may cause risks to you. If you are already enrolled in another research study, please inform Dr. Wendy Dahl listed in question 3 of this consent form) or the person reviewing this consent with you before enrolling in this or any other research study or project.
Throughout the study, the researchers will notify you of new information that may become available and might affect your decision to remain in the study.
If you wish to discuss the information above or any discomforts you may experience, please ask questions now or call the PI or contact person listed on the front page of this form.
Other possible risks to you may include: none
Researchers will take appropriate steps to protect any information they collect about you. However, there is a slight risk that information about you could be revealed inappropriately or accidentally. Depending on the nature of the information, such a release could upset or embarrass you, or possibly affect your insurability or employability. Questions 17-21 in this form discuss what information about you will be collected, used, protected, and shared.
This study may include risks that are unknown at this time.
Participation in more than one research study or project may further increase the risks to you. If you are already enrolled in another research study, please inform one of the research team members listed in question 3 of this form or the person reviewing this consent with you before enrolling in this or any other research study or project.
Throughout the study, the researchers will notify you of new information that may become available and might affect your decision to remain in the study.
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If you wish to discuss the information above or any discomforts you may experience, please ask questions now or call one of the research team members listed in question 3 in this form.
11a. What are the potential benefits to you for taking part in this research study?
There is no direct benefit to you for participating in this research study.
11b. How could others possibly benefit from this study?
Research into the efficacy of probiotic survival may lead to a better understanding of B. subtilis as a single probiotic agent.
11c. How could the researchers benefit from this study?
In general, presenting research results helps the career of a scientist. Therefore, the Principal Investigator listed in question 3 of this form may benefit if the results of this study are presented at scientific meetings or in scientific journals.
12. What other choices do you have if you do not want to be in this study?
You have the option to not take part in this study. If you do not want to take part in this study tell the Principle Investigator and do not sign this consent form.
13a. Can you withdraw from this study?
You are free to withdraw your consent and to stop participating in this study at any time. If you do withdraw your consent, you will not be penalized in any way and you will not lose any benefits to which you are entitled.
If you decide to withdraw your consent to participate in this study for any reason, please contact one of the research team members listed in question 3 of this form. They will tell you how to stop your participation safely.
If you have any questions regarding your rights as a research subject, please call the Institutional Review Board (IRB) office at (352) 273-9600.
13b. If you withdraw, can information about you still be used and/or collected?
If you withdraw your consent, your information will not be used.
13c. Can the Principal Investigator withdraw you from this study?
You may be withdrawn from the study without your consent for the following reasons:
You did not follow the instructions given, e.g. did not consume the probiotics or complete the daily diary.
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WHAT ARE THE FINANCIAL ISSUES IF YOU PARTICIPATE?
14. If you choose to take part in this research study, will it cost you anything? The sponsor will only pay for medical services that you receive as part of your participation in this study as described in question 7 above. All other medical services that you would have received if you were not in this study will be billed to you or your insurance company. You will be responsible for paying any deductible, co-insurance, co-payments for those services and for any non-covered or out –of –network services. Some insurance companies may not cover costs associated with studies. Please contact your insurance company for additional information. The probiotic B. subtilis, questionnaires, and sample containers will be provided at no cost to you while you are participating in this study. The Sponsor will pay for medical services or activities required as part of your participation in this study. There will be no cost to you. If you receive a bill related to this study, please contact Wendy Dahl, 352 392 1991 ext 224. All other medical services provided to you that are not directly related to the study will be billed to you or your insurance company. You will be responsible for paying any deductible, co-insurance, and/or co-payments for these services, and any non-covered or out-of-network services.
Some insurance companies may not cover costs associated with studies. Please contact your insurance company for additional information.
15. Will you be paid for taking part in this study?
Yes, you will be paid $15 per stool and $155 for taking the capsules and completing the questionnaires for a maximum total of $200 for participating in the entire study. If you are paid for taking part in this study, your name and social security number will be reported to the appropriate University employees for purposes of making and recording the payment. You are responsible for paying income taxes on any payments provided by the study. If the payments total $600 or more, the University must report the amount you received to the Internal Revenue Service (IRS).
16. What if you are injured because of the study?
If you are injured as a direct result of your participation in this study, only the professional services that you receive from any University of Florida Health Science Center healthcare provider will be provided without charge. These healthcare providers include physicians,
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physician assistants, nurse practitioners, dentists or psychologists. Any other expenses, including Shands hospital expenses, will be billed to you or your insurance provider.
You will be responsible for any deductible, co-insurance, or co-payments. Some insurance companies may not cover costs associated with research studies or research-related injuries. Please contact your insurance company for additional information.
Please contact one of the research team members listed in question 3 of this form if you experience an injury or have questions about any discomforts that you experience while participating in this study.
17. How will your health information be collected, used and shared?
If you agree to participate in this study, the Principal Investigator will create, collect, and use private information about you and your health. This information is called protected health information or PHI. In order to do this, the Principal Investigator needs your authorization The following section describes what PHI will be collected, used and shared, how it will be collected, used, and shared, who will collect, use or share it, who will have access to it, how it will be secured, and what your rights are to revoke this authorization.
Your protected health information may be collected, used, and shared with others to determine if you can participate in the study, and then as part of your participation in the study. This information can be gathered from you or your past, current or future health records, from procedures such as physical examinations, x-rays, blood or urine tests or from other procedures or tests. This information will be created by receiving study treatments or participating in study procedures, or from your study visits and telephone calls. More specifically, the following information may be collected, used, and shared with others:
Name
Address
Phone Number
Your social security number for compensation purposes
Body weight
Height
Age
Sex
Gastrointestinal wellness and symptoms
Stool microbiota data
This information will be stored in locked filing cabinets or on computer servers with secure passwords, or encrypted electronic storage devices.
Some of the information collected could be included in a "limited data set" to be used for other research purposes. If so, the limited data set will only include information that does not directly identify you. For example, the limited data set cannot include your name, address, telephone number, social security number, photographs, or other codes that link you to the information in the limited data set. If limited data sets are created and used,
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agreements between the parties creating and receiving the limited data set are required in order to protect your identity and confidentiality and privacy.
18. For what study-related purposes will your protected health information be collected, used, and shared with others?
Your PHI may be collected, used, and shared with others to make sure you can participate in the research, through your participation in the research, and to evaluate the results of the research study. More specifically, your PHI may be collected, used, and shared with others for the following study-related purpose(s): To determine the survival of three doses of B. subtilis delivered in capsules on transit survival, impact on the intestinal microbiome, gastrointestinal symptoms and general well-being in healthy human adults age 18 to 50.
Once this information is collected, it becomes part of the research record for this study.
19. Who will be allowed to collect, use, and share your protected health information?
Only certain people have the legal right to collect, use and share your research records, and they will protect the privacy and security of these records to the extent the law allows. These people include:
The study Principal Investigator (listed in question 3 of this form) and research staff associated with this project.
Other professionals at the University of Florida or Shands Hospital that provide study-related treatment or procedures.
The University of Florida Institutional Review Board (IRB; an IRB is a group of people who are responsible for looking after the rights and welfare of people taking part in research).
20. Once collected or used, who may your protected health information be shared with?
Your PHI may be shared with:
The study sponsor (listed in Question 4 of this form).
United States and foreign governmental agencies who are responsible for overseeing research, such as the Food and Drug Administration, the Department of Health and Human Services, and the Office of Human Research Protections .
Government agencies who are responsible for overseeing public health concerns such as the Centers for Disease Control and federal, state and local health departments.
Otherwise, your research records will not be released without your permission unless required by law or a court order. It is possible that once this information is shared with
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authorized persons, it could be shared by the persons or agencies who receive it and it would no longer be protected by the federal medical privacy law.
21. If you agree to take part in this research study, how long will your protected health information be used and shared with others?
Your PHI will be used and shared with others for one year following the completion of the study.
You are not required to sign this consent and authorization or allow researchers to collect, use and share your PHI. Your refusal to sign will not affect your treatment, payment, enrollment, or eligibility for any benefits outside this research study. However, you cannot participate in this research unless you allow the collection, use and sharing of your protected health information by signing this consent and authorization.
You have the right to review and copy your protected health information. However, we can make this available only after the study is finished.
You can revoke your authorization at any time before, during, or after your participation in this study. If you revoke it, no new information will be collected about you. However, information that was already collected may still be used and shared with others if the researchers have relied on it to complete the research. You can revoke your authorization by giving a written request with your signature on it to the Principal Investigator.
SIGNATURES
As an investigator or the investigator’s representative, I have explained to the participant the purpose, the procedures, the possible benefits, and the risks of this research study; the alternative to being in the study; and how the participant’s protected health information will be collected, used, and shared with others:
Signature of Person Obtaining Consent and Authorization
Date
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You have been informed about this study’s purpose, procedures, possible benefits, and risks; the alternatives to being in the study; and how your protected health information will be collected, used and shared with others. You have received a copy of this Form. You have been given the opportunity to ask questions before you sign, and you have been told that you can ask questions at any time. You voluntarily agree to participate in this study. You hereby authorize the collection, use and sharing of your protected health information as described in sections 17-21 above. By signing this form, you are not waiving any of your legal rights.
Signature of Person Consenting and Authorizing Date
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APPENDIX C GLOBAL PHYSICAL ACTIVITY QUESTIONNNAIRE
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APPENDIX D DAILY QUESTIONNAIRE
Did you experience any of the following symptoms in the last 24 hours? Please circle one choice 0-10, 0= none, 3= mild, 6= very severe. Note: If you are a female, please do not rate menstrual cramping and bloating. Please rate the severity by circling the appropriate number below Bloating 0 1 2 3 4 5 6 None moderate very severe Flatulence 0 1 2 3 4 5 6 None moderate very severe Abdominal Cramping 0 1 2 3 4 5 6 None moderate very severe Stomach Noises 0 1 2 3 4 5 6 None moderate very severe Headache 0 1 2 3 4 5 6 None moderate very severe Dizziness
0 1 2 3 4 5 6 None moderate very severe Sore throat 0 1 2 3 4 5 6 None moderate very severe
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Blocked ear canal 0 1 2 3 4 5 6 None moderate very severe Nasal congestion
0 1 2 3 4 5 6 None moderate very severe Runny eyes 0 1 2 3 4 5 6 None moderate very severe Nausea
0 1 2 3 4 5 6 None moderate very severe Vomiting
0 1 2 3 4 5 6 None moderate very severe Diarrhea
0 1 2 3 4 5 6 None moderate very severe Feeling of constipation
0 1 2 3 4 5 6 None moderate very severe Feeling of satiety (not hungry)
0 1 2 3 4 5 6 None moderate very severe Itching
0 1 2 3 4 5 6 None moderate very severe
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Fatigue 0 1 2 3 4 5 6 None moderate very severe Skin rash
0 1 2 3 4 5 6 None moderate very severe Skin redness/flushing
0 1 2 3 4 5 6 None moderate very severe Feelings of anxiety
0 1 2 3 4 5 6 None moderate very severe Feelings of depression 0 1 2 3 4 5 6 None moderate very severe How many stools (bowel movements) did you have today? ______________
0 1 2 3 4 5 6 7 8 9 10 >10 What is your energy level today?
Excellent Very good Good Fair Poor
How many hours did you sleep last night? Do not include the time it took you to fall
asleep or anytime you were awakened during the night. Circle the time that most closely matches your sleep.
<5 hours 5-6 hours 6-7 hours 7-8 hours 8-9 hours >9 hours
Did you take your probiotic capsule today? Yes No Did you visit a doctor today? Yes No
Did you take an antibiotic today? Yes No
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APPENDIX E GASTROINTESTINAL SYMPTOM RESPONSE SCALE
(1) No discomfort at all (5) Moderately severe discomfort (2) Slight discomfort (6) Severe discomfort (3) Mild discomfort (7) Very severe discomfort (4) Moderate discomfort Questions: Answer each question using the response scale above.
_____Have you been bothered by stomach ache or pain during the past week?
(Stomach ache refers to all kinds of aches or pains in your stomach or belly) _____Have you been bothered by heartburn during the past week? (By heartburn we mean a burning pain or discomfort behind the breastbone in your chest) _____Have you been bothered by acid reflux during the past week? (By acid reflux we mean regurgitation or flow of sour or bitter fluid into your mouth) _____Have you been bothered by hunger pains in the stomach or belly during the
past week? (This hallow feeling in the stomach is associated with the need to eat between meals.) _____Have you been bothered by nausea during the past week?
(By nausea we mean a feeling of wanting to be sick) _____Have you been bothered by rumbling in your stomach or belly during the past week? (Rumbling refers to vibrations or noises in the stomach) _____Has your stomach felt boated during the past week? (Feeling bloated refers to swelling in the stomach or belly) _____Have you been bothered by burping during the past week?
(Burping refers to bringing up air or gas through the mouth) _____Have you been bothered by passing gas or flatus during the past week? (Passing gas refers to the release of air or gas from the bowels) _____Have you been bothered by constipation during the past week?
(Constipation refers to a reduced ability to empty the bowels) _____Have you been bothered by diarrhea during the past week? (Diarrhea refers to frequent loose or watery stools) _____Have you been bothered by loose stools during the past week?
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(If your stools have been alternately hard and loose, this question only refers to the extent you have been bothered by the stools being loose) _____Have you been bothered by hard stools during the past week? (If your stools have been alternately hard and loose, this question only refers to the extent you have been bothered by stools being hard) _____Have you been bothered by an urgent need to have a bowel movement during the past week? (This urgent need to open your bowels makes you rush to the toilet) _____When going to the toilet during the past week, have you had the feeling of not
completely emptying your bowels? (The feeling that after finishing a bowel
movement, there is still some stool that needs to be passed)
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BIOGRAPHICAL SKETCH
Abdulah Hanifi was born in Castro Valley, California. The youngest of four
children, he grew up mostly in Clovis, California, graduating from University High School
in 2007. He attended California State University Fresno and earned his B.A. in
philosophy with an emphasis in religious studies, and chemistry minor in 2011. He
graduated magna cum laude and was awarded a Dean’s Medal for outstanding
academic achievement in the College of Arts and Humanities. His involvement at the
University of California San Francisco-Fresno Department of Emergency Medicine’s
Academic Research Associate Program afforded him the valuable opportunity to work
closely with physicians and medical staff conducting clinical research on emergency
room patients.
His training in the humanities, sciences and clinical research allowed him the
opportunity to begin his M.S. in food science and human nutrition at the University of
Florida. He began his work studying the clinical outcomes on the consumption of a
novel probiotic in 2012 and upon completion of his M.S. program, Abdulah will continue
his studies to earn a Ph.D. in food science and human nutrition.